Question: do individuals who recover from COVID-19 show sufficient immune response, including antibody levels and T-cell mediated immunity, to prevent re-infection?

Title: Characterization of anti-viral immunity in recovered individuals infected by SARS-CoV-2 Content: The WHO has declared SARS-CoV-2 outbreak a public health emergency of international concern. However, to date, there was hardly any study in characterizing the immune responses, especially adaptive immune responses to SARS-CoV-2 infection. In this study, we collected blood from COVID-19 patients who have recently become virus-free and therefore were discharged, and analyzed their SARS-CoV-2-specific antibody and T cell responses. We observed SARS-CoV-2-specific humoral and cellular immunity in the patients. Both were detected in newly discharged patients, suggesting both participate in immune-mediated protection to viral infection. However, follow-up patients (2 weeks post discharge) exhibited high titers of IgG antibodies, but with low levels of virus-specific T cells, suggesting that they may enter a quiescent state. Our work has thus provided a basis for further analysis of protective immunity to SARS-CoV-2, and understanding the pathogenesis of COVID-19, especially in the severe cases. It has also implications in designing an effective vaccine to protect and treat SARS-CoV-2 infection.

Title: T-cell-mediated immune response to respiratory coronaviruses Content: Emerging respiratory coronaviruses such as the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and Middle East Respiratory Syndrome coronavirus (MERS-CoV) pose potential biological threats to humans. SARS and MERS are manifested as severe atypical pneumonia associated with high morbidity and mortality in humans. The majority of studies carried out in SARS-CoV-infected humans and animals attribute a dysregulated/exuberant innate response as a leading contributor to SARS-CoV-mediated pathology. A decade after the 2002–2003 SARS epidemic, we do not have any approved preventive or therapeutic agents available in case of re-emergence of SARS-CoV or other related viruses. A strong neutralizing antibody response generated against the spike (S) glycoprotein of SARS-CoV is completely protective in the susceptible host. However, neutralizing antibody titers and the memory B cell response are short-lived in SARS-recovered patients and the antibody will target primary homologous strain. Interestingly, the acute phase of SARS in humans is associated with a severe reduction in the number of T cells in the blood. Surprisingly, only a limited number of studies have explored the role of the T cell-mediated adaptive immune response in respiratory coronavirus pathogenesis. In this review, we discuss the role of anti-virus CD4 and CD8 T cells during respiratory coronavirus infections with a special emphasis on emerging coronaviruses.

Title: The underpinning biology relating to multiple sclerosis disease modifying treatments during the COVID-19 pandemic Content: BACKGROUND: SARS-CoV-2 viral infection causes COVID-19 that can result in severe acute respiratory distress syndrome (ARDS), which can cause significant mortality, leading to concern that immunosuppressive treatments for multiple sclerosis and other disorders have significant risks for both infection and ARDS. OBJECTIVE: To examine the biology that potentially underpins immunity to the SARS-Cov-2 virus and the immunity-induced pathology related to COVID-19 and determine how this impinges on the use of current disease modifying treatments in multiple sclerosis. OBSERVATIONS: Although information about the mechanisms of immunity are scant, it appears that monocyte/macrophages and then CD8 T cells are important in eliminating the SARS-CoV-2 virus. This may be facilitated via anti-viral antibody responses that may prevent re-infection. However, viral escape and infection of leucocytes to promote lymphopenia, apparent CD8 T cell exhaustion coupled with a cytokine storm and vascular pathology appears to contribute to the damage in ARDS. IMPLICATIONS: In contrast to ablative haematopoietic stem cell therapy, most multiple-sclerosis-related disease modifying therapies do not particularly target the innate immune system and few have any major long-term impact on CD8 T cells to limit protection against COVID-19. In addition, few block the formation of immature B cells within lymphoid tissue that will provide antibody-mediated protection from (re)infection. However, adjustments to dosing schedules may help de-risk the chance of infection further and reduce the concerns of people with MS being treated during the COVID-19 pandemic.

Title: The underpinning biology relating to multiple sclerosis disease modifying treatments during the COVID-19 pandemic. Content: BACKGROUND: : SARS-CoV-2 viral infection causes COVID-19 that can result in severe acute respiratory distress syndrome (ARDS), which can cause significant mortality, leading to concern that immunosuppressive treatments for multiple sclerosis and other disorders have significant risks for both infection and ARDS. OBJECTIVE: : To examine the biology that potentially underpins immunity to the SARS-Cov-2 virus and the immunity-induced pathology related to COVID-19 and determine how this impinges on the use of current disease modifying treatments in multiple sclerosis. OBSERVATIONS: : Although information about the mechanisms of immunity are scant, it appears that monocyte/macrophages and then CD8 T cells are important in eliminating the SARS-CoV-2 virus. This may be facilitated via anti-viral antibody responses that may prevent re-infection. However, viral escape and infection of leucocytes to promote lymphopenia, apparent CD8 T cell exhaustion coupled with a cytokine storm appears to contribute to the damage in ARDS. IMPLICATIONS: : In contrast to ablative haematopoietic stem cell therapy, most multiple-sclerosis-related disease modifying therapies do not particularly target the innate immune system and few have any major long-term impact on CD8 T cells to limit protection against COVID-19. In addition, few block the formation of immature B cells within lymphoid tissue that will provide antibody-mediated protection from (re)infection. However, adjustments to dosing schedules may help de-risk the chance of infection further and reduce the concerns of people with MS being treated during the COVID-19 pandemic.

Title: Humoral and circulating follicular helper T cell responses in recovered patients with COVID-19 Content: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has dramatically expedited global vaccine development efforts1–3, most targeting the viral ‘spike’ glycoprotein (S) S localizes on the virion surface and mediates recognition of cellular receptor angiotensin-converting enzyme 2 (ACE2)4–6 Eliciting neutralizing antibodies that block S–ACE2 interaction7–9, or indirectly prevent membrane fusion10, constitute an attractive modality for vaccine-elicited protection11 However, although prototypic S-based vaccines show promise in animal models12–14, the immunogenic properties of S in humans are poorly resolved In this study, we characterized humoral and circulating follicular helper T cell (cTFH) immunity against spike in recovered patients with coronavirus disease 2019 (COVID-19) We found that S-specific antibodies, memory B cells and cTFH are consistently elicited after SARS-CoV-2 infection, demarking robust humoral immunity and positively associated with plasma neutralizing activity Comparatively low frequencies of B cells or cTFH specific for the receptor binding domain of S were elicited Notably, the phenotype of S-specific cTFH differentiated subjects with potent neutralizing responses, providing a potential biomarker of potency for S-based vaccines entering the clinic Overall, although patients who recovered from COVID-19 displayed multiple hallmarks of effective immune recognition of S, the wide spectrum of neutralizing activity observed suggests that vaccines might require strategies to selectively target the most potent neutralizing epitopes

Title: Virus-specific memory CD8 T cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection. Content: UNLABELLED Severe acute respiratory syndrome coronavirus (SARS-CoV) caused an acute human respiratory illness with high morbidity and mortality in 2002-2003. Several studies have demonstrated the role of neutralizing antibodies induced by the spike (S) glycoprotein in protecting susceptible hosts from lethal infection. However, the anti-SARS-CoV antibody response is short-lived in patients who have recovered from SARS, making it critical to develop additional vaccine strategies. SARS-CoV-specific memory CD8 T cells persisted for up to 6 years after SARS-CoV infection, a time at which memory B cells and antivirus antibodies were undetectable in individuals who had recovered from SARS. In this study, we assessed the ability of virus-specific memory CD8 T cells to mediate protection against infection in the absence of SARS-CoV-specific memory CD4 T or B cells. We demonstrate that memory CD8 T cells specific for a single immunodominant epitope (S436 or S525) substantially protected 8- to 10-month-old mice from lethal SARS-CoV infection. Intravenous immunization with peptide-loaded dendritic cells (DCs) followed by intranasal boosting with recombinant vaccinia virus (rVV) encoding S436 or S525 resulted in accumulation of virus-specific memory CD8 T cells in bronchoalveolar lavage fluid (BAL), lungs, and spleen. Upon challenge with a lethal dose of SARS-CoV, virus-specific memory CD8 T cells efficiently produced multiple effector cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukin 2 [IL-2]) and cytolytic molecules (granzyme B) and reduced lung viral loads. Overall, our results show that SARS-CoV-specific memory CD8 T cells protect susceptible hosts from lethal SARS-CoV infection, but they also suggest that SARS-CoV-specific CD4 T cell and antibody responses are necessary for complete protection. IMPORTANCE Virus-specific CD8 T cells are required for pathogen clearance following primary SARS-CoV infection. However, the role of SARS-CoV-specific memory CD8 T cells in mediating protection after SARS-CoV challenge has not been previously investigated. In this study, using a prime-boost immunization approach, we showed that virus-specific CD8 T cells protect susceptible 8- to 10-month-old mice from lethal SARS-CoV challenge. Thus, future vaccines against emerging coronaviruses should emphasize the generation of a memory CD8 T cell response for optimal protection.

Title: Natural T Cell-mediated Protection against Seasonal and Pandemic Influenza. Results of the Flu Watch Cohort Study. Content: RATIONALE A high proportion of influenza infections are asymptomatic. Animal and human challenge studies and observational studies suggest T cells protect against disease among those infected, but the impact of T-cell immunity at the population level is unknown. OBJECTIVES To investigate whether naturally preexisting T-cell responses targeting highly conserved internal influenza proteins could provide cross-protective immunity against pandemic and seasonal influenza. METHODS We quantified influenza A(H3N2) virus-specific T cells in a population cohort during seasonal and pandemic periods between 2006 and 2010. Follow-up included paired serology, symptom reporting, and polymerase chain reaction (PCR) investigation of symptomatic cases. MEASUREMENTS AND MAIN RESULTS A total of 1,414 unvaccinated individuals had baseline T-cell measurements (1,703 participant observation sets). T-cell responses to A(H3N2) virus nucleoprotein (NP) dominated and strongly cross-reacted with A(H1N1)pdm09 NP (P < 0.001) in participants lacking antibody to A(H1N1)pdm09. Comparison of paired preseason and post-season sera (1,431 sets) showed 205 (14%) had evidence of infection based on fourfold influenza antibody titer rises. The presence of NP-specific T cells before exposure to virus correlated with less symptomatic, PCR-positive influenza A (overall adjusted odds ratio, 0.27; 95% confidence interval, 0.11-0.68; P = 0.005, during pandemic [P = 0.047] and seasonal [P = 0.049] periods). Protection was independent of baseline antibodies. Influenza-specific T-cell responses were detected in 43%, indicating a substantial population impact. CONCLUSIONS Naturally occurring cross-protective T-cell immunity protects against symptomatic PCR-confirmed disease in those with evidence of infection and helps to explain why many infections do not cause symptoms. Vaccines stimulating T cells may provide important cross-protective immunity.

Title: Intrafamilial Exposure to SARS-CoV-2 Induces Cellular Immune Response without Seroconversion Content: Background. In the background of the current COVID-19 pandemic, serological tests are being used to assess past infection and immunity against SARS-CoV-2. This knowledge is paramount to determine the transmission dynamics of SARS-CoV-2 through the post pandemic period. Several individuals belonging to households with an index COVID-19 patient, reported symptoms of COVID-19 but discrepant serology results. Methods. Here we investigated the humoral and cellular immune responses against SARS-CoV-2 in seven families, including nine index patients and eight contacts, who had evidence of serological discordances within the households. Ten unexposed healthy donors were enrolled as controls. Results. All index patients recovered from a mild COVID-19. They all developed anti-SARS-CoV-2 antibodies and a significant T cell response detectable up to 69 days after symptom onset. Six of the eight contacts reported COVID-19 symptoms within 1 to 7 days after the index patients but all were SARS-CoV-2 seronegative. Six out of eight contacts developed a SARS-CoV-2-specific T cell response against structural and/or accessory proteins that lasts up to 80 days post symptom onset suggesting a past SARS-CoV-2 infection. Conclusion. Exposure to SARS-CoV-2 can induce virus-specific T cell responses without seroconversion. T cell responses may be more sensitive indicators of SARS-Co-V-2 exposure than antibodies. Our results indicate that epidemiological data relying only on the detection of SARS-CoV-2 antibodies may lead to a substantial underestimation of prior exposure to the virus

Title: Immune responses to porcine epidemic diarrhea virus (PEDV) in swine and protection against subsequent infection Content: Understanding the immune responses against Porcine epidemic diarrhea virus (PEDV) is important to prevent infection and to design control strategies. We evaluated both systemic and mucosal immune responses to PEDV in pigs and assessed if prior exposure to virus protects against re-infection. Three-week-old pigs were infected with PEDV and immune response in blood, intestine, and mesenteric lymph node (MLN) was evaluated. At 30 dpi, virus exposed pigs were challenged with a field isolate of PEDV and immune response at 5 d post challenge was evaluated. We found that PEDV RNA persists in the intestine even after fecal shedding of the virus was stopped at 28 dpi and pigs previously exposed to PEDV are protected from virus shedding after re-infection. PEDV infection induced both humoral and cell mediated immune response with an increase in PEDV specific IgA and IgG antibodies in intestine and serum. Flow cytometry analysis showed a significantly higher frequency of B cells and lower frequency of T cells at 4 dpi. The frequency of CD4/CD8 double positive (DP) memory T cells was significantly increased in the MLN of challenged animals. These studies may provide further insights into understanding the mucosal immune response to PEDV and its role in protection against disease.

Title: Generation of protective pneumococcal-specific nasal resident memory CD4(+) T cells via parenteral immunization Content: The generation of tissue-resident memory T cells (T(RM)) is an essential aspect of immunity at mucosal surfaces, and it has been suggested that preferential generation of T(RM) is one of the principal advantages of mucosally administered vaccines. We have previously shown that antigen-specific, IL-17-producing CD4(+) T cells can provide capsular antibody-independent protection against nasal carriage of Streptococcus pneumoniae; but whether pneumococcus-responsive T(RM) are localized within the nasal mucosa and are sufficient for protection from carriage has not been determined. Here we show that intranasal administration of live or killed pneumococci to mice generates pneumococcus-responsive IL-17A-producing CD4(+) mucosal T(RM). Furthermore, we show that these cells are sufficient to mediate long-lived, neutrophil-dependent protection against subsequent pneumococcal nasal challenge. Unexpectedly, and in contrast with the prevailing paradigm, we found that parenteral administration of killed pneumococci also generates protective IL-17A(+)CD4(+) T(RM) in the nasal mucosa. These results demonstrate a critical and sufficient role of T(RM) in prevention of pneumococcal colonization, and further that these cells can be generated by parenteral immunization. Our findings therefore have important implications regarding the generation of immune protection at mucosal surfaces by vaccination.

Title: Characterization of SARS-CoV-specific memory T cells from recovered individuals 4 years after infection Content: SARS-CoV infection of human results in antigen-specific cellular and humoral immune responses. However, it is critical to determine whether SARS-CoV-specific memory T cells can persist for long periods of time. In this study, we analyzed the cellular immune response from 21 SARS-recovered individuals who had been diagnosed with SARS in 2003 by using ELISA, CBA, ELISpot and multiparameter flow cytometry assays. Our results demonstrated that low levels of specific memory T cell responses to SARS-CoV S, M, E and N peptides were detected in a proportion of SARS-recovered patients, and IFN-γ was the predominant cytokine produced by T cells after stimulation with peptides. Cytometry analysis indicated that the majority of memory CD8(+) T cells produced IFN-γ, whereas memory CD4(+) T cells produced IFN-γ, IL-2 or TNF-α. These results might provide valuable information on the cellular immune response in recovered SARS-CoV patients for the rational design of vaccines against SARS-CoV infection.

Title: COVID-19 and the immune system. Content: A close interaction between the virus SARS-CoV-2 and the immune system of an individual results in a diverse clinical manifestation of the COVID-19 disease. While adaptive immune responses are essential for SARS-CoV-2 virus clearance, the innate immune cells, such as macrophages, may contribute, in some cases, to the disease progression. Macrophages have shown a significant production of IL-6 suggesting they may contribute to the excessive inflammation in COVID-19 disease. Macrophage Activation Syndrome may further explain the high serum levels of CRP, which are normally lacking in viral infections. In adaptive immune responses, it has been revealed that cytotoxic CD8+ T cells exhibit functional exhaustion patterns, such as the expression of NKG2A, PD-1, and TIM-3. Since SARS-CoV-2 restrains antigen presentation by downregulating MHC class I and II molecules and, therefore, inhibits the T cell-mediated immune responses, humoral immune responses also play a substantial role. Specific IgA response appears to be stronger and more persistent than IgM response. Moreover, IgM and IgG antibodies show similar dynamics in COVID-19 disease.

Title: COVID-19 and the immune system Content: A close interaction between the virus SARS-CoV-2 and the immune system of an individual results in a diverse clinical manifestation of the COVID-19 disease While adaptive immune responses are essential for SARS-CoV-2 virus clearance, the innate immune cells, such as macrophages, may contribute, in some cases, to the disease progression Macrophages have shown a significant production of IL-6 suggesting they may contribute to the excessive inflammation in COVID-19 disease Macrophage Activation Syndrome may further explain the high serum levels of CRP, which are normally lacking in viral infections In adaptive immune responses, it has been revealed that cytotoxic CD8+ T cells exhibit functional exhaustion patterns, such as the expression of NKG2A, PD-1, and TIM-3 Since SARS-CoV-2 restrains antigen presentation by downregulating MHC class I and II molecules and, therefore, inhibits the T cell-mediated immune responses, humoral immune responses also play a substantial role Specific IgA response appears to be stronger and more persistent than IgM response Moreover, IgM and IgG antibodies show similar dynamics in COVID-19 disease

Title: VCAM-1/α4β1 integrin interaction is crucial for prompt recruitment of immune T cells into the brain during the early stage of reactivation of chronic infection with Toxoplasma gondii to prevent toxoplasmic encephalitis. Content: Reactivation of chronic infection with Toxoplasma gondii can cause life-threatening toxoplasmic encephalitis in immunocompromised individuals. We examined the role of VCAM-1/α4β1 integrin interaction in T cell recruitment to prevent reactivation of the infection in the brain. SCID mice were infected and treated with sulfadiazine to establish a chronic infection. VCAM-1 and ICAM-1 were the endothelial adhesion molecules detected on cerebral vessels of the infected SCID and wild-type animals. Immune T cells from infected wild-type mice were treated with anti-α4 integrin or control antibodies and transferred into infected SCID or nude mice, and the animals received the same antibody every other day. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Expression of mRNAs for CD3δ, CD4, CD8β, gamma interferon (IFN-γ), and inducible nitric oxide synthase (NOS2) (an effector molecule to inhibit T. gondii growth) and the numbers of CD4(+) and CD8(+) T cells in the brain were significantly less in mice treated with anti-α4 integrin antibody than in those treated with control antibody at 3 days after sulfadiazine discontinuation. At 6 days after sulfadiazine discontinuation, cerebral tachyzoite-specific SAG1 mRNA levels and numbers of inflammatory foci associated with tachyzoites were markedly greater in anti-α4 integrin antibody-treated than in control antibody-treated animals, even though IFN-γ and NOS2 mRNA levels were higher in the former than in the latter. These results indicate that VCAM-1/α4β1 integrin interaction is crucial for prompt recruitment of immune T cells and induction of IFN-γ-mediated protective immune responses during the early stage of reactivation of chronic T. gondii infection to control tachyzoite growth.

Title: Hyperactive immune cells (T cells) may be responsible for acute lung injury in influenza virus infections: A need for early immune-modulators for severe cases Content: It has been believed that acute lung injury in influenza virus infections is caused by a virus-induced cytopathy; viruses that have multiplied in the upper respiratory tract spread to lung tissues along the lower respiratory tract. However, some experimental and clinical studies have suggested that the pathogenesis of acute lung injury in influenza virus infections is associated with excessive host response including a cell-mediated immune reaction. During the pandemic H1N1 2009 influenza A virus infections in Korea, we experienced a dramatic effect of immune-modulators (corticosteroids) on the patients with severe pneumonia who had significant respiratory distress at presentation and those who showed rapidly progressive pneumonia during oseltamivir treatment. We also found that the pneumonia patients treated with corticosteroids showed the lowest lymphocyte differential and that the severity of pneumonia was associated with the lymphocyte count at presentation. From our findings and previous experimental and clinical studies, we postulated that hyperactive immune cells (T cells) may be involved in the acute lung injury of influenza virus infections, using a hypothesis of ‘protein homeostasis system’; the inducers of the cell-mediated immune response are initially produced at the primary immune sites by the innate immune system. These substances reach the lung cells, the main target organ, via the systemic circulation, and possibly the cells of other organs, including myocytes or central nerve system cells, leading to extrapulmonary symptoms (e.g., myalgia and rhabdomyolysis, and encephalopathy). To control these substances that may be possibly toxic to host cells, the adaptive immune reaction may be operated by immune cells, mainly lymphocytes. Hyperimmune reaction of immune cells produces higher levels of cytokines which may be associated with acute lung injury, and may be controlled by early use of immune-modulators. Early initiation and proper dosage of immune-modulators with antiviral agents for severe pneumonia patients may reduce morbidity and prevent progressive fatal pneumonia.

Title: Memory CD8 T cells mediate severe immunopathology following respiratory syncytial virus infection Content: Memory CD8 T cells can provide protection from re-infection by respiratory viruses such as influenza and SARS. However, the relative contribution of memory CD8 T cells in providing protection against respiratory syncytial virus (RSV) infection is currently unclear. To address this knowledge gap, we utilized a prime-boost immunization approach to induce robust memory CD8 T cell responses in the absence of RSV-specific CD4 T cells and antibodies. Unexpectedly, RSV infection of mice with pre-existing CD8 T cell memory led to exacerbated weight loss, pulmonary disease, and lethal immunopathology. The exacerbated disease in immunized mice was not epitope-dependent and occurred despite a significant reduction in RSV viral titers. In addition, the lethal immunopathology was unique to the context of an RSV infection as mice were protected from a normally lethal challenge with a recombinant influenza virus expressing an RSV epitope. Memory CD8 T cells rapidly produced IFN-γ following RSV infection resulting in elevated protein levels in the lung and periphery. Neutralization of IFN-γ in the respiratory tract reduced morbidity and prevented mortality. These results demonstrate that in contrast to other respiratory viruses, RSV-specific memory CD8 T cells can induce lethal immunopathology despite mediating enhanced viral clearance.

Title: The immune response to influenza in older humans: beyond immune senescence Content: Despite widespread influenza vaccination programs, influenza remains a major cause of morbidity and mortality in older adults. Age-related changes in multiple aspects of the adaptive immune response to influenza have been well-documented including a decline in antibody responses to influenza vaccination and changes in the cell-mediated response associated with immune senescence. This review will focus on T cell responses to influenza and influenza vaccination in older adults, and how increasing frailty or coexistence of multiple (≥2) chronic conditions contributes to the loss of vaccine effectiveness for the prevention of hospitalization. Further, dysregulation of the production of pro- and anti-inflammatory mediators contributes to a decline in the generation of an effective CD8 T cell response needed to clear influenza virus from the lungs. Current influenza vaccines provide only a weak stimulus to this arm of the adaptive immune response and rely on re-stimulation of CD8 T cell memory related to prior exposure to influenza virus. Efforts to improve vaccine effectiveness in older adults will be fruitless until CD8 responses take center stage.

Title: Long-Term Persistence of Robust Antibody and Cytotoxic T Cell Responses in Recovered Patients Infected with SARS Coronavirus Content: Most of the individuals infected with SARS coronavirus (SARS-CoV) spontaneously recovered without clinical intervention. However, the immunological correlates associated with patients' recovery are currently unknown. In this report, we have sequentially monitored 30 recovered patients over a two-year period to characterize temporal changes in SARS-CoV-specific antibody responses as well as cytotoxic T cell (CTL) responses. We have found persistence of robust antibody and CTL responses in all of the study subjects throughout the study period, with a moderate decline one year after the onset of symptoms. We have also identified two potential major CTL epitopes in N proteins based on ELISPOT analysis of pooled peptides. However, despite the potent immune responses and clinical recovery, peripheral lymphocyte counts in the recovered patients have not yet been restored to normal levels. In summary, our study has, for the first time, characterized the temporal and dynamic changes of humoral and CTL responses in the natural history of SARS-recovered individuals, and strongly supports the notion that high and sustainable levels of immune responses correlate strongly with the disease outcome. Our findings have direct implications for future design and development of effective therapeutic agents and vaccines against SARS-CoV infection.

Title: Immune responses and pathogenesis of SARS-CoV-2 during an outbreak in Iran: Comparison with SARS and MERS Content: The beginning of 2020 has seen the emergence of COVID-19, an outbreak caused by a novel coronavirus, SARS-CoV-2, an important pathogen for humans. There is an urgent need to better understand this new virus and to develop ways to control its spread. In Iran, the first case of the COVID-19 was reported after spread from China and other countries. Fever, cough, and fatigue were the most common symptoms of this virus. In worldwide, the incubation period of COVID-19 was 3 to 7 days and approximately 80% of infections are mild or asymptomatic, 15% are severe, requiring oxygen, and 5% are critical infections, requiring ventilation. To mount an antiviral response, the innate immune system recognizes molecular structures that are produced by the invasion of the virus. COVID-19 infection induces IgG antibodies against N protein that can be detected by serum as early as day 4 after the onset of disease and with most patients seroconverting by day 14. Laboratory evidence of clinical patients showed that a specific T-cell response against SARS-CoV-2 is important for the recognition and killing of infected cells, particularly in the lungs of infected individuals. At present, there is no specific antiviral therapy for COVID-19 and the main treatments are supportive. In this review, we investigated the innate and acquired immune responses in patients who recovered from COVID-19, which could inform the design of prophylactic vaccines and immunotherapy for the future.

Title: Immune responses and pathogenesis of SARS‐CoV‐2 during an outbreak in Iran: Comparison with SARS and MERS Content: The beginning of 2020 has seen the emergence of COVID‐19, an outbreak caused by a novel coronavirus, SARS‐CoV‐2, an important pathogen for humans. There is an urgent need to better understand this new virus and to develop ways to control its spread. In Iran, the first case of the COVID‐19 was reported after spread from China and other countries. Fever, cough, and fatigue were the most common symptoms of this virus. In worldwide, the incubation period of COVID‐19 was 3 to 7 days and approximately 80% of infections are mild or asymptomatic, 15% are severe, requiring oxygen, and 5% are critical infections, requiring ventilation. To mount an antiviral response, the innate immune system recognizes molecular structures that are produced by the invasion of the virus. COVID‐19 infection induces IgG antibodies against N protein that can be detected by serum as early as day 4 after the onset of disease and with most patients seroconverting by day 14. Laboratory evidence of clinical patients showed that a specific T‐cell response against SARS‐CoV‐2 is important for the recognition and killing of infected cells, particularly in the lungs of infected individuals. At present, there is no specific antiviral therapy for COVID‐19 and the main treatments are supportive. In this review, we investigated the innate and acquired immune responses in patients who recovered from COVID‐19, which could inform the design of prophylactic vaccines and immunotherapy for the future.

Title: T-cell epitopes in severe acute respiratory syndrome (SARS) coronavirus spike protein elicit a specific T-cell immune response in patients who recover from SARS. Content: The immunogenicity of HLA-A2-restricted T-cell epitopes in the S protein of the Severe acute respiratory syndrome coronavirus (SARS-CoV) and of human coronavirus strain 229e (HCoV-229e) was analyzed for the elicitation of a T-cell immune response in donors who had fully recovered from SARS-CoV infection. We employed online database analysis to compare the differences in the amino acid sequences of the homologous T epitopes of HCoV-229e and SARS-CoV. The identified T-cell epitope peptides were synthesized, and their binding affinities for HLA-A2 were validated and compared in the T2 cell system. The immunogenicity of all these peptides was assessed by using T cells obtained from donors who had fully recovered from SARS-CoV infection and from healthy donors with no history of SARS-CoV infection. HLA-A2 typing by indirect immunofluorescent antibody staining showed that 51.6% of SARS-CoV-infected patients were HLA-A2 positive. Online database analysis and the T2 cell binding test disclosed that the number of HLA-A2-restricted immunogenic epitopes of the S protein of SARS-CoV was decreased or even lost in comparison with the homologous sequences of the S protein of HCoV-229e. Among the peptides used in the study, the affinity of peptides from HCoV-229e (H77 and H881) and peptides from SARS-CoV (S978 and S1203) for binding to HLA-A2 was higher than that of other sequences. The gamma interferon (IFN-gamma) release Elispot assay revealed that only SARS-CoV-specific peptides S1203 and S978 induced a high frequency of IFN-gamma-secreting T-cell response in HLA-A2(+) donors who had fully recovered from SARS-CoV infection; such a T-cell epitope-specific response was not observed in HLA-A2(+) healthy donors or in HLA-A2(-) donors who had been infected with SARS-CoV after full recovery. Thus, T-cell epitopes S1203 and S978 are immunogenic and elicit an overt specific T-cell response in HLA-A2(+) SARS-CoV-infected patients.

Title: Deregulated cellular circuits driving immunoglobulins and complement consumption associate with the severity of COVID-19 Content: Background: SARS-CoV-2 infection causes an abrupt response by the host immune system, which is largely responsible for the pathogenesis and outcome of COVID-19. We aimed to investigate which specific responses from either cellular or humoral immunity associate to severity and progression of COVID-19. Methods: A cohort of 276 patients classified in mild, moderate and severe, was studied. Peripheral blood lymphocyte subpopulations were quantified by flow cytometry, and immunoglobulins and complement proteins by nephelometry. Results: At admission, dramatic lymphopenia of T, B and NK cells associated to severity. However, only the proportion of B cells increased, while T and NK cells appeared unaffected. Accordingly, the number of plasma cells and circulating follicular helper T cells (cTfh) increased, but levels of IgM, IgA and IgG were unaffected. When degrees of severity were considered, IgG was lower in severe patients, suggesting an IgG consumption by complement activation or antibody-dependent cellular cytotoxicity (ADCC). Activated CD56-CD16+ NK-cells, which mediate ADCC, were increased. Regarding complement, C3 and C4 protein levels were higher in mild and moderate, but not in severe patients, compared to healthy donors. Moreover, IgG and C4 decreased from day 0 to day 10 in patients who were hospitalized for more than two weeks, but not in patients who were discharged earlier. Conclusion: Our study provides important clues to understand the immune response observed in COVID-19 patients, which is probably related to viral clearance, but also underlies its pathogenesis and severity. This study associates for the first time COVID-19 severity with an imbalanced humoral immune response characterized by excessive consumption of IgG and C4, identifying new targets for therapeutic intervention.

Title: Comparative evaluation of two hemagglutinating encephalomyelitis coronavirus vaccine candidates in mice. Content: Porcine hemagglutinating encephalomyelitis (PHE) is caused by the coronavirus hemagglutinating encephalomyelitis virus (PHE-CoV), and the recent, rapid spread of PHE-CoV in piglets from many countries emphasizes the urgent need for a PHE-CoV vaccine. Here we use a murine model for evaluation of the induction of humoral and cellular immune responses by inactivated and PHE-CoV DNA vaccines in order to define the immune correlates for protection against PHE-CoV. The inactivated vaccine was composed of purified PHE-CoV and aluminum hydroxide gel (alum), which was chosen as an adjuvant because of its long history of safety for human use. The PHE-CoV DNA vaccine was constructed by subcloning the S1 gene of PHE-CoV into the pVAX1 vector to create the recombinant plasmid pV-S1. Our results showed that the inactivated PHE-CoV vaccine (IPV) elicited a high level of humoral immunity, resulting in good protection efficacy against PHE-CoV challenge. The IPV induced the IgG1 subclass of serum antibodies and expression of the cytokine interleukin-4 (IL-4), suggesting that the IPV generated a predominantly Th2-type immune response. The DNA vaccine was found to mediate primarily a cellular immune response with high levels of IgG2a and the cytokines IL-2 and gamma interferon (IFN-γ). However, mice that were vaccinated twice with the DNA vaccine and boosted with the IPV could mount a sufficient neutralizing antibody response against live PHE-CoV, with little variation in IgG1 and IgG2a levels, and showed high levels of IL-2 and IL-4. This response may activate both B and T cells to mount a specific humoral and cellular immune response that could, in turn, elicit a phagocyte-mediated defense against PHE-CoV infections to achieve viral clearance.

Title: Phagocytic cells contribute to the antibody-mediated elimination of pulmonary-infected SARS coronavirus Content: Abstract While the 2002–2003 outbreak of severe acute respiratory syndrome (SARS) resulted in 774 deaths, patients who were affected with mild pulmonary symptoms successfully recovered. The objective of the present work was to identify, using SARS coronavirus (SARS-CoV) mouse infection models, immune factors responsible for clearing of the virus. The elimination of pulmonary SARS-CoV infection required the activation of B cells by CD4+ T cells. Furthermore, passive immunization (post-infection) with homologous (murine) anti-SARS-CoV antiserum showed greater elimination efficacy against SARS-CoV than that with heterologous (rabbit) antiserum, despite the use of equivalent titers of neutralizing antibodies. This distinction was mediated by mouse phagocytic cells (monocyte-derived infiltrating macrophages and partially alveolar macrophages, but not neutrophils), as demonstrated both by adoptive transfer from donors and by immunological depletion of selected cell types. These results indicate that the cooperation of anti-SARS-CoV antibodies and phagocytic cells plays an important role in the elimination of SARS-CoV.

Title: Rapid expansion of CD8+ T cells in wild-type and type I interferon receptor-deficient mice correlates with protection after low-dose emergency immunization with modified vaccinia virus Ankara. Content: UNLABELLED Immunization with modified vaccinia virus Ankara (MVA) can rapidly protect mice against lethal ectromelia virus (ECTV) infection, serving as an experimental model for severe systemic infections. Importantly, this early protective capacity of MVA vaccination completely depends on virus-specific cytotoxic CD8(+) T cell responses. We used MVA vaccination in the mousepox challenge model using ECTV infection to investigate the previously unknown factors required to elicit rapid protective T cell immunity in normal C57BL/6 mice and in mice lacking the interferon alpha/beta receptor (IFNAR(-/-)). We found a minimal dose of 10(5) PFU of MVA vaccine fully sufficient to allow robust protection against lethal mousepox, as assessed by the absence of disease symptoms and failure to detect ECTV in organs from vaccinated animals. Moreover, MVA immunization at low dosage also protected IFNAR(-/-) mice, indicating efficient activation of cellular immunity even in the absence of type I interferon signaling. When monitoring for virus-specific CD8(+) T cell responses in mice vaccinated with the minimal protective dose of MVA, we found significantly enhanced levels of antigen-specific T cells in animals that were MVA vaccinated and ECTV challenged compared to mice that were only vaccinated. The initial priming of naive CD8(+) T cells by MVA immunization appears to be highly efficient and, even at low doses, mediates a rapid in vivo burst of pathogen-specific T cells upon challenge. Our findings define striking requirements for protective emergency immunization against severe systemic infections with orthopoxviruses. IMPORTANCE We demonstrate that single-shot low-dose immunizations with vaccinia virus MVA can rapidly induce T cell-mediated protective immunity against lethal orthopoxvirus infections. Our data provide new evidence for an efficient protective capacity of vaccination with replication-deficient MVA. These data are of important practical relevance for public health, as the effectiveness of a safety-tested, next-generation smallpox vaccine based on MVA is still debated. Furthermore, producing sufficient amounts of vaccine is expected to be a major challenge should an outbreak occur. Moreover, prevention of other infections may require rapidly protective immunization; hence, MVA could be an extremely useful vaccine for delivering heterologous T cell antigens, particularly for infectious diseases that fit a scenario of emergency vaccination.

Title: Immunity to Infection Content: This chapter describes immune responses to the six major types of pathogens: extracellular bacteria, intracellular bacteria, viruses, parasites, fungi and prions. Innate immunity mediated by neutrophils, NK cells, NKT cells, γδ T cells, complement and microbicidal molecules prevents infection or slows it until adaptive immunity can also respond to the pathogen. Extracellular entities are coated in antibody and cleared by antibody- and complement-mediated mechanisms. Parasitic worms are prevented from anchoring in the host by IgA and IgE antibodies. IgE triggers mast cell, basophil and eosinophil degranulation and the release of toxic mediators. Intracellular bacteria and parasites as well as viruses are eliminated by CTLs, NK cells, NKT cells and γδ T cells secreting cytotoxic cytokines and/or carrying out target cell cytolysis. Macrophage hyperactivation and granuloma formation may be triggered to confine persistent invaders. Th1 and Th17 responses support cell-mediated immunity against internal threats, whereas Th2 responses support humoral immunity against external threats. Each type of pathogen has evolved to evade immune responses by avoiding recognition or inactivating various leukocyte effector mechanisms.

Title: Increased expression of CD8 marker on T-cells in COVID-19 patients Content: Abstract Background Cell-mediated immunity including T-cells (T helper and cytotoxic) plays an essential role in efficient antiviral responses against coronavirus disease-2019 (COVID-19). Therefore, in this study, we evaluated the ratio and expression of CD4 and CD8 markers in COVID-19 patients to clarify the immune characterizations of CD4 and CD8 T-cells in COVID-19 patients. Methods Peripheral blood samples of 25 COVID-19 patients and 25 normal individuals with similar age and sex as the control group were collected. White blood cells, platelets, and lymphocytes were counted and CD4 and CD8 T lymphocytes were evaluated by flow cytometry. Results The number of white blood cells, lymphocytes, and platelets were reduced significantly in COVID-19 patients (P < 0.05). The difference in CD4:CD8 ratio, CD4 T-cell frequency, CD8 T-cell frequency, and CD4 mean fluorescence intensity (MFI) was not significant between COVID-19 patients and healthy individuals (P > 0.05); however, the CD8 MFI increased significantly in COVID-19 infected patients (P < 0.05). Conclusion Although, there is no significant difference in the ratio of CD4 to CD8 between two groups, the expression level of CD8 in COVID-19 patients was significantly higher than the normal individuals. This result suggested that the cellular immune responses triggered by COVID-19 infection were developed through overexpression of CD8 and hyperactivation of cytotoxic T lymphocytes.

Title: Increased expression of CD8 marker on T-cells in COVID-19 patients Content: BACKGROUND: Cell-mediated immunity including T-cells (T helper and cytotoxic) plays an essential role in efficient antiviral responses against coronavirus disease-2019 (COVID-19). Therefore, in this study, we evaluated the ratio and expression of CD4 and CD8 markers in COVID-19 patients to clarify the immune characterizations of CD4 and CD8 T-cells in COVID-19 patients. METHODS: Peripheral blood samples of 25 COVID-19 patients and 25 normal individuals with similar age and sex as the control group were collected. White blood cells, platelets, and lymphocytes were counted and CD4 and CD8 T lymphocytes were evaluated by flow cytometry. RESULTS: The number of white blood cells, lymphocytes, and platelets were reduced significantly in COVID-19 patients (P < 0.05). The difference in CD4:CD8 ratio, CD4 T-cell frequency, CD8 T-cell frequency, and CD4 mean fluorescence intensity (MFI) was not significant between COVID-19 patients and healthy individuals (P > 0.05); however, the CD8 MFI increased significantly in COVID-19 infected patients (P < 0.05). CONCLUSION: Although, there is no significant difference in the ratio of CD4 to CD8 between two groups, the expression level of CD8 in COVID-19 patients was significantly higher than the normal individuals. This result suggested that the cellular immune responses triggered by COVID-19 infection were developed through overexpression of CD8 and hyperactivation of cytotoxic T lymphocytes.

Title: Induction of mucosal immune responses and protection against enteric viruses: rotavirus infection of gnotobiotic pigs as a model Content: Abstract Enteric viruses are a major cause of diarrhea in animals and humans. Among them, rotaviruses are one of the most important causes of diarrhea in young animals and human infants. A lack of understanding of mechanisms to induce intestinal immunity and the correlates of protective immunity in neonates has impaired development of safe and effective vaccines against enteric viruses. Studies of candidate vaccines using an adult mouse model of subclinical enteric viral infections often do not predict vaccine efficacy against disease evaluated in neonatal large animals. A series of studies have been conducted using a neonatal gnotobiotic pig model of rotavirus infection and diarrhea to identify correlates of protective immunity and to evaluate traditional and novel vaccine approaches for the induction of mucosal immune responses and protection to enteric viruses. Gnotobiotic pigs recovered from infection with virulent Wa human rotavirus (HRV) (mimic natural infection) had high numbers of intestinal IgA rotavirus-specific primary antibody-secreting cells (ASCs) and memory B-cells (to recall antigen) measured by ELISPOT assay, which correlated with complete protection against rotavirus challenge. Most short-term IgA memory B-cells were resident in the ileum, the major site of rotavirus replication. Spleen, not the bone marrow, was the major resident site for longer-term IgG memory B-cells. Candidate rotavirus vaccines evaluated in pigs for their ability to induce intestinal or systemic ASC and protection against rotavirus infection and diarrhea included attenuated live virus, inactivated virus, and baculovirus-expressed double-layered rotavirus-like particles (2/6-VLPs). In combination with those candidate vaccines, various adjuvants, delivery systems, and immunization routes were tested, including incomplete Freund’s adjuvant for i.m. immunization, and a mutant Escherichia coli heat labile enterotoxin R192G (mLT) for i.n. immunization. It was shown that orally administered replicating vaccines were most effective for priming for intestinal IgA ASC and memory B-cell responses, but i.n. administered non-replicating 2/6-VLPs plus mLT were effective as booster vaccines. We conclude that protective immunity depends on the magnitude, location, viral protein-specificity, and isotype of the antibody responses induced by vaccination. Therefore highly effective enteric viral vaccines should: (i) induce sufficient levels of intestinal IgA antibodies; (ii) include viral antigens that induce neutralizing antibodies; and (iii) require the use of effective mucosal adjuvants or antigen delivery systems for non-replicating oral or i.n. vaccines.

Title: Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19 Content: SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. We systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in a large cohort of unexposed individuals as well as exposed family members and individuals with acute or convalescent COVID-19. Acute phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative family members and individuals with a history of asymptomatic or mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits robust memory T cell responses akin to those observed in the context of successful vaccines, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19 also in seronegative individuals.

Title: Personalized workflow to identify optimal T-cell epitopes for peptide-based vaccines against COVID-19 Content: Traditional vaccines against viruses are designed to target their surface proteins, i.e., antigens, which can trigger the immune system to produce specific antibodies to capture and neutralize the viruses. However, viruses often evolve quickly, and their antigens are prone to mutations to avoid recognition by the antibodies (antigenic drift). This limitation of the antibody-mediated immunity could be addressed by the T-cell mediated immunity, which is able to recognize conserved viral HLA peptides presented on virus-infected cells. Thus, by targeting conserved regions on the genome of a virus, T-cell epitope-based vaccines are less subjected to mutations and may work effectively on different strains of the virus. Here we propose a personalized workflow to identify an optimal set of T-cell epitopes based on the HLA alleles and the immunopeptidome of an individual person. Specifically, our workflow trains a machine learning model on the immunopeptidome and then predicts HLA peptides from conserved regions of a virus that are most likely to trigger responses from the person T cells. We applied the workflow to identify T-cell epitopes for the SARS-COV-2 virus, which has caused the recent COVID-19 pandemic in more than 100 countries across the globe.

Title: Innate Immunity in Viral Encephalitis Content: Innate immune responses to pathogens are evolutionarily ancient and are found in the most primitive organisms. These are highly conserved and are not pathogen-specific, but are in response to classes of molecular structures. Infections can be perceived both extracellularly and intracellularly by Pathogen Associated Molecular Patterns (PAMPs) and their host cell ligands, Pathogen Recognition Receptors (PRRs), among them, Toll-Like Receptors (TLRs). The innate immune response to infection includes the release of soluble preformed mediators, or synthesis of cytoplasmic enzymes, cytokines, chemokines, interferons (IFNs), lipid mediators, proteins of the complement cascade, neurotransmitters, nucleotides, and components of transcription factors (High Mobility Group B1, receptors for sex hormones/steroids). Directed cellular migration of parenchymal astrocytes and microglia, as well as recruitment across the blood brain barrier (BBB) of circulating neutrophils, natural killer, monocytes, macrophages, dendritic cells, and ultimately T lymphocytes to the site of infection are also hallmarks of innate responses to infections. These responding cells contribute their own secreted effector molecules and effector activities (such as phagocytosis). Distinct viruses are capable of infecting every cell type (endothelial cells, ependymal cells, perivascular macrophages and pericytes, astrocytes, microglia, oligodendrocytes, Schwann cells, and neurons) in the central nervous system (CNS). These CNS infections challenge the host with a different set of problems than do peripheral viral infections. Among the complications are (a) neurons that rarely express Class I or Class II Major Histocompatibility Complex (MHC) molecules and are thus not suitable targets for either CD4(+) or CD8(+) MHC-restricted T cells, (b) an enclosed volume that is constrained from swelling during inflammation, as well as poorly developed lymphatic drainage, and (c) the immunologic privilege of the CNS which leads to extremely limited immune surveillance for pathogens. Therefore, the role of innate immunity, both from CNS-resident cells and their products, and from circulating inflammatory cells and molecules which traverse the BBB are essential to “buy time,” inhibiting viral replication and dissemination, until the host can marshal an adaptive immune response. The immune responses are crucial for host survival from the infection. Consequently, successful pathogens, especially those that persist, have developed a wide variety of evasive approaches to limit the inhibition of replication. Many of these pathways are highlighted in individual chapters that precede this one. These evasive measures range from neutralizing host secreted molecules (cytokines and chemokines) with soluble receptors, encoding anti-inflammatory proteins in their genome, preventing signal transduction, blocking inhibition of protein synthesis, degradation of essential antiviral molecules, preventing apoptosis, and blockade of the nuclear pore complex. In this chapter, I will attempt to cover the breadth of the innate immune response to viral infection, but will also devote more space to generally under-considered aspects than to the well-known components. There are some caveats to consider, as most experiments have been performed in the murine model and not in man; further, conclusions from experiments using in vitro cultured cells (whether primary or established lines) may not reflect physiological conditions in an undisturbed CNS. Lastly, we now appreciate the complexities imposed on hosts by polymorphisms in genes of critical pathways, leading to increased susceptibility or resistance of that individual but not others.

Title: Different pattern of pre-existing SARS-COV-2 specific T cell immunity in SARS-recovered and uninfected individuals Content: Memory T cells induced by previous infections can influence the course of new viral infections. Little is known about the pattern of SARS-CoV-2 specific pre-existing memory T cells in human. Here, we first studied T cell responses to structural (nucleocapsid protein, NP) and non-structural (NSP-7 and NSP13 of ORF1) regions of SARS-CoV-2 in convalescent from COVID-19 (n=24). In all of them we demonstrated the presence of CD4 and CD8 T cells recognizing multiple regions of the NP protein. We then show that SARS-recovered patients (n=23), 17 years after the 2003 outbreak, still possess long-lasting memory T cells reactive to SARS-NP, which displayed robust cross-reactivity to SARS-CoV-2 NP. Surprisingly, we observed a differential pattern of SARS-CoV-2 specific T cell immunodominance in individuals with no history of SARS, COVID-19 or contact with SARS/COVID-19 patients (n=18). Half of them (9/18) possess T cells targeting the ORF-1 coded proteins NSP7 and 13, which were rarely detected in COVID-19- and SARS-recovered patients. Epitope characterization of NSP7-specific T cells showed recognition of protein fragments with low homology to “common cold” human coronaviruses but conserved among animal betacoranaviruses. Thus, infection with betacoronaviruses induces strong and long-lasting T cell immunity to the structural protein NP. Understanding how pre-existing ORF-1-specific T cells present in the general population impact susceptibility and pathogenesis of SARS-CoV-2 infection is of paramount importance for the management of the current COVID-19 pandemic.

Title: Could Intravenous Immunoglobulin Collected from Recovered Coronavirus Patients Protect against COVID-19 and Strengthen the Immune System of New Patients? Content: The emergence of the novel coronavirus in Wuhan, China, which causes severe respiratory tract infections in humans (COVID-19), has become a global health concern Most coronaviruses infect animals but can evolve into strains that cross the species barrier and infect humans At the present, there is no single specific vaccine or efficient antiviral therapy against COVID-19 Recently, we showed that intravenous immunoglobulin (IVIg) treatment reduces inflammation of intestinal epithelial cells and eliminates overgrowth of the opportunistic human fungal pathogen Candida albicans in the murine gut Immunotherapy with IVIg could be employed to neutralize COVID-19 However, the efficacy of IVIg would be better if the immune IgG antibodies were collected from patients who have recovered from COVID-19 in the same city, or the surrounding area, in order to increase the chance of neutralizing the virus These immune IgG antibodies will be specific against COVID-19 by boosting the immune response in newly infected patients Different procedures may be used to remove or inactivate any possible pathogens from the plasma of recovered coronavirus patient derived immune IgG, including solvent/detergent, 60 °C heat-treatment, and nanofiltration Overall, immunotherapy with immune IgG antibodies combined with antiviral drugs may be an alternative treatment against COVID-19 until stronger options such as vaccines are available

Title: Could Intravenous Immunoglobulin Collected from Recovered Coronavirus Patients Protect against COVID-19 and Strengthen the Immune System of New Patients? Content: The emergence of the novel coronavirus in Wuhan, China, which causes severe respiratory tract infections in humans (COVID-19), has become a global health concern. Most coronaviruses infect animals but can evolve into strains that cross the species barrier and infect humans. At the present, there is no single specific vaccine or efficient antiviral therapy against COVID-19. Recently, we showed that intravenous immunoglobulin (IVIg) treatment reduces inflammation of intestinal epithelial cells and eliminates overgrowth of the opportunistic human fungal pathogen Candida albicans in the murine gut. Immunotherapy with IVIg could be employed to neutralize COVID-19. However, the efficacy of IVIg would be better if the immune IgG antibodies were collected from patients who have recovered from COVID-19 in the same city, or the surrounding area, in order to increase the chance of neutralizing the virus. These immune IgG antibodies will be specific against COVID-19 by boosting the immune response in newly infected patients. Different procedures may be used to remove or inactivate any possible pathogens from the plasma of recovered coronavirus patient derived immune IgG, including solvent/detergent, 60 °C heat-treatment, and nanofiltration. Overall, immunotherapy with immune IgG antibodies combined with antiviral drugs may be an alternative treatment against COVID-19 until stronger options such as vaccines are available.

Title: Dynamics and Differences in Systemic and Local Immune Responses After Vaccination With Inactivated and Live Commercial Vaccines and Subsequent Subclinical Infection With PRRS Virus Content: The goals of our study were to compare the immune response to different killed and modified live vaccines against PRRS virus and to monitor the antibody production and the cell mediated immunity both at the systemic and local level. In the experiment, we immunized four groups of piglets with two commercial inactivated (A1—Progressis, A2—Suivac) and two modified live vaccines (B3—Amervac, B4—Porcilis). Twenty-one days after the final vaccination, all piglets, including the control non-immunized group (C5), were i.n., infected with the Lelystad strain of PRRS virus. The serum antibody response (IgM and IgG) was the strongest in group A1 followed by two MLV (B3 and B4) groups. Locally, we demonstrated the highest level of IgG antibodies in bronchoalveolar lavages (BALF), and saliva in group A1, whereas low IgA antibody responses in BALF and feces were detected in all groups. We have found virus neutralization antibody at DPV 21 (days post vaccination) and higher levels in all groups including the control at DPI 21 (days post infection). Positive antigen specific cell-mediated response in lymphocyte transformation test (LTT) was observed in groups B3 and B4 at DPV 7 and in group B4 at DPV 21 and in all intervals after infection. The IFN-γ producing lymphocytes after antigen stimulation were found in CD4(−)CD8(+) and CD4(+)CD8(+) subsets of all immunized groups 7 days after infection. After infection, there were obvious differences in virus excretion. The virus was detected in all groups of piglets in serum, saliva, and occasionally in feces at DPI 3. Significantly lower virus load was found in groups A1 and B3 at DPI 21. Negative samples appeared at DPI 21 in B3 group in saliva. It can be concluded that antibodies after immunization and infection, and the virus after infection can be detected in all the compartments monitored. Immunization with inactivated vaccine A1—Progressis induces high levels of antibodies produced both systemically and locally. Immunization with MLV-vaccines (Amervac and Porcilis) produces sufficient antibody levels and also cell-mediated immunity. After infection virus secretion gradually decreases in group B3, indicating tendency to induce sterile immunity.

Title: A possible role of immunopathogenesis in COVID-19 progression Content: Background: The efficacy of the humoral and cellular immunity determines the outcome of viral infections. An appropriate immune response mediates protection, whereas an overwhelming immune response has been associated with immune-mediated pathogenesis in viral infections. The current study explored the general and SARS-CoV-2 specific cellular and humoral immune status in patients with different COVID-19 severities. Methods: In this prospective study, we included 53 patients with moderate, severe, and critical COVID-19 manifestations comparing their quantitative, phenotypic, and functional characteristics of circulating immune cells, SARS-CoV-2 antigen specific T-cells, and humoral immunity. Results: Significantly diminished frequencies of CD8+T-cells, CD4+ and CD8+T-cell subsets with activated differentiated memory/effector phenotype and migratory capacity were found in circulation in patients with severe and/or critical COVID-19 as compared to patients with moderate disease. Importantly, the improvement of the clinical courses from severe to moderate was accompanied by an improvement in the T-cell subset alterations. Furthermore, we surprisingly observed a detectable SARS-CoV-2-reactive T-cell response in all three groups after stimulation with SARS-CoV-2 S-protein overlapping peptide pool already at the first visit. Of note, patients with a critical COVID-19 demonstrated a stronger response of SARS-CoV-2-reactive T-cells producing Th1 associated inflammatory cytokines. Furthermore, clear correlation between antibody titers and SARS-CoV-2-reactive CD4+ frequencies underscore the role of specific immunity in disease progression. Conclusion: Our data demonstrate that depletion of activated memory phenotype circulating T-cells and a strong SARS-CoV-2-specific cellular and humoral immunity are associated with COVID-19 disease severity. This counter-intuitive finding may have important implications for diagnostic, therapeutic and prophylactic COVID-19 management.

Title: Lack of cytomegalovirus (CMV)-specific cell-mediated immune response using QuantiFERON-CMV assay in CMV-seropositive healthy volunteers: fact not artifact. Content: The QuantiFERON-CMV (QF) assay measures cell-mediated immunity against cytomegalovirus (CMV-CMI), which is particularly useful in individuals susceptible to CMV infection such as transplant patients. A positive QF result identifies patients that are better protected against CMV infection. However, the significance of a negative QF result in CMV-seropositive individuals needs to be clarified. CMV-CMI was analyzed in healthy subjects using the QF assay, and, in parallel, the Flow-cytometric Assay of Specific Cell-mediated Immune response in Activated whole blood (FASCIA). FASCIA assay measures T-cell proliferation using CMV lysate as stimulus whereas QF assay use a mix of peptides. A total of 93 healthy volunteers were enrolled, and 13/71 CMV-seropositive individuals (18.3%) showed humoral/cellular discordance using QF assay (CMV+ QF-). Interestingly, with FASCIA assay CD4+ and CD8+ T-cell proliferations were lower in CMV+ QF- than in CMV+ QF+ individuals. Furthermore, CMV+ QF- volunteers had a lower level of anti-CMV IgG than CMV+ QF+ subjects. Discordant CMV+ QF- volunteers can be defined as low responder individuals since they show lower CMV-specific humoral and cellular immune responses in comparison to CMV+ QF+ individuals. Immune discordance shows the high heterogeneity of immunity to CMV in healthy subjects.

Title: Lack of cytomegalovirus (CMV)-specific cell-mediated immune response using QuantiFERON-CMV assay in CMV-seropositive healthy volunteers: fact not artifact Content: The QuantiFERON-CMV (QF) assay measures cell-mediated immunity against cytomegalovirus (CMV-CMI), which is particularly useful in individuals susceptible to CMV infection such as transplant patients. A positive QF result identifies patients that are better protected against CMV infection. However, the significance of a negative QF result in CMV-seropositive individuals needs to be clarified. CMV-CMI was analyzed in healthy subjects using the QF assay, and, in parallel, the Flow-cytometric Assay of Specific Cell-mediated Immune response in Activated whole blood (FASCIA). FASCIA assay measures T-cell proliferation using CMV lysate as stimulus whereas QF assay use a mix of peptides. A total of 93 healthy volunteers were enrolled, and 13/71 CMV-seropositive individuals (18.3%) showed humoral/cellular discordance using QF assay (CMV+ QF-). Interestingly, with FASCIA assay CD4+ and CD8+ T-cell proliferations were lower in CMV+ QF- than in CMV+ QF+ individuals. Furthermore, CMV+ QF- volunteers had a lower level of anti-CMV IgG than CMV+ QF+ subjects. Discordant CMV+ QF- volunteers can be defined as low responder individuals since they show lower CMV-specific humoral and cellular immune responses in comparison to CMV+ QF+ individuals. Immune discordance shows the high heterogeneity of immunity to CMV in healthy subjects.

Title: Antibody-driven design of a human cytomegalovirus gHgLpUL128L subunit vaccine that selectively elicits potent neutralizing antibodies. Content: The use of neutralizing antibodies to identify the most effective antigen has been proposed as a strategy to design vaccines capable of eliciting protective B-cell immunity. In this study, we analyzed the human antibody response to cytomegalovirus (human cytomegalovirus, HCMV) infection and found that antibodies to glycoprotein (g)B, a surface glycoprotein that has been developed as a HCMV vaccine, were primarily nonneutralizing. In contrast, most of the antibodies to the complex formed by gH, gL, protein (p)UL128, pUL130, and pUL131 (the gHgLpUL128L pentamer) neutralized HCMV infection with high potency. Based on this analysis, we developed a single polycistronic vector encoding the five pentamer genes separated by "self-cleaving" 2A peptides to generate a stably transfected CHO cell line constitutively secreting high levels of recombinant pentamer that displayed the functional antigenic sites targeted by human neutralizing antibodies. Immunization of mice with the pentamer formulated with different adjuvants elicited HCMV neutralizing antibody titers that persisted to high levels over time and that were a hundred- to thousand-fold higher than those found in individuals that recovered from primary HCMV infection. Sera from mice immunized with the pentamer vaccine neutralized infection of both epithelial cells and fibroblasts and prevented cell-to-cell spread and viral dissemination from endothelial cells to leukocytes. Neutralizing monoclonal antibodies from immunized mice showed the same potency as human antibodies and targeted the same as well as additional sites on the pentamer. These results illustrate with a relevant example a general and practical approach of analytic vaccinology for the development of subunit vaccines against complex pathogens.

Title: The art of survival during viral persistence. Content: Central nervous system infection by the neurotropic JHM strain of mouse hepatitis virus (JHMV) results in chronic demyelination characterized by viral persistence in the absence of infectious virus. CD8(+) T cells inhibit acute viral replication via cell type-specific effector mechanisms. Perforin-mediated cytolysis controls virus in microglia/macrophages and astrocytes, whereas interferon (IFN)-gamma regulates viral replication in oligodendroglia. JHMV infection of antibody-deficient mice confirmed a primary role of cellular immunity and a redundant role for humoral immunity during acute infection. However, infectious virus reactivates in antibody-deficient mice following viral clearance. This observation suggests that virus-specific T cells in the central nervous system are unable to control viral persistence. Reactivation in antibody-deficient mice is not associated with increased T-cell infiltration, but is prevented via transfer of neutralizing antibody. A vital role for humoral immunity during persistence is supported by the accumulation and retention of virus-specific antibody secreting cells following clearance of infectious virus. Thus, cell-mediated immune responses control acute infection, whereas humoral immunity maintains viral persistence. Therefore, although the central nervous system provides an environment for prolonged retention of both T cells and plasma cells, plasma cells are critical in maintaining persistent virus at undetectable levels. The low turnover of virus, T cells, and B cells constitute a unifying feature of persistent infection, illustrating the dichotomy between distinct immune effectors in regulating acute and persistent central nervous system infection.

Title: SARS-CoV-2 activates lung epithelia cell proinflammatory signaling and leads to immune dysregulation in COVID-19 patients by single-cell sequencing Content: Objective: The outbreak of Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection has become a global health emergency. We aim to decipher SARS-CoV-2 infected cell types, the consequent host immune response and their interplay in the lung of COVID-19 patients. Design: We analyzed single-cell RNA sequencing (scRNA-seq) data of lung samples from 17 subjects (6 severe COVID-19 patients, 3 mild patients who recovered and 8 healthy controls). The expression of SARS-CoV-2 receptors (ACE2 and TMPRSS2) was examined among different cell types in the lung. The immune cells infiltration patterns, their gene expression profiles, and the interplay of immune cells and SARS-CoV-2 target cells were further investigated. Results: Compared to healthy controls, the overall ACE2 (receptor of SARS-CoV-2) expression was significantly higher in lung epithelial cells of COVID-19 patients, in particular in ciliated cell, club cell and basal cell. Comparative transcriptome analysis of these lung epithelial cells of COVID-19 patients and healthy controls identified that SARS-CoV-2 infection activated pro-inflammatory signaling including interferon pathway and cytokine signaling. Moreover, we identified dysregulation of immune response in patients with COVID-19. In severe COVID-19 patients, significantly higher neutrophil, but lower T and NK cells in lung were observed along with markedly increased cytokines (CCL2, CCL3, CCL4, CCL7, CCL3L1 and CCL4L2) compared with healthy controls as well as mild patients who recovered. The cytotoxic phenotypes were shown in lung T and NK cells of severe patients as evidenced by enhanced IFN{gamma}, Granulysin, Granzyme B and Perforin expression. Moreover, SARS-CoV-2 infection altered the community interplay of lung epithelial cells and immune cells: the interaction between epithelial cells with macrophage, T and NK cell was stronger, but their interaction with neutrophils was lost in COVID-19 patients compared to healthy controls. Conclusions: SARS-CoV-2 infection activates pro-inflammatory signaling in lung epithelial cells expressing ACE2 and causes dysregulation of immune response to release more pro-inflammatory cytokines. Moreover, SARS-CoV-2 infection breaks the interplay of lung epithelial cells and immune cells.

Title: Innate immunity in COVID-19 patients mediated by NKG2A receptors, and potential treatment using Monalizumab, Cholroquine, and antiviral agents Content: Following the outbreak of a novel coronavirus (SARS-CoV-2), studies suggest that the resultant disease (COVID-19) is more severe in individuals with a weakened immune system. Cytotoxic T-cells (CTLs) and Natural Killer (NK) cells are required to generate an effective immune response against viruses, functional exhaustion of which enables disease progression. Patients with severe COVID-19 present significantly lower lymphocyte, and higher neutrophil, counts in blood. Specifically, CD8+ lymphocytes and NK cells were significantly reduced in cases of severe infection compared to patients with mild infection and healthy individuals. The NK group 2 member A (NKG2A) receptor transduces inhibitory signalling, suppressing NK cytokine secretion and cytotoxicity. Overexpression of NKG2A has been observed on CD8+ and NK cells of COVID-19 infected patients compared to healthy controls, while NKG2A overexpression also functionally exhausts CD8+ cells and NK cells, resulting in a severely compromised innate immune response. Blocking NKG2A on CD8+ cells and NK cells in cancers modulated tumor growth, restoring CD8+ T and NK cell function. A recently proposed mechanism via which SARS-CoV-2 overrides innate immune response of the host is by over-expressing NKG2A on CD+ T and NK cells, culminating in functional exhaustion of the immune response against the viral pathogen. Monalizumab is an inhibiting antibody against NKG2A which can restore the function of CD8 + T and NK cells in cancers, successfully ceasing tumor progression with no significant side effects in Phase 2 clinical trials. We hypothesize that patients with severe COVID-19 have a severely compromised innate immune response and could be treated via the use of Monalizumab, interferon α, chloroquine, and other antiviral agents.

Title: Innate immunity in COVID-19 patients mediated by NKG2A receptors, and potential treatment using Monalizumab, Cholroquine, and antiviral agents Content: Abstract Following the outbreak of a novel coronavirus (SARS-CoV-2), studies suggest that the resultant disease (COVID-19) is more severe in individuals with a weakened immune system. Cytotoxic T-cells (CTLs) and Natural Killer (NK) cells are required to generate an effective immune response against viruses, functional exhaustion of which enables disease progression. Patients with severe COVID-19 present significantly lower lymphocyte, and higher neutrophil, counts in blood. Specifically, CD8+ lymphocytes and NK cells were significantly reduced in cases of severe infection compared to patients with mild infection and healthy individuals. The NK group 2 member A (NKG2A) receptor transduces inhibitory signalling, suppressing NK cytokine secretion and cytotoxicity. Overexpression of NKG2A has been observed on CD8+ and NK cells of COVID-19 infected patients compared to healthy controls, while NKG2A overexpression also functionally exhausts CD8+ cells and NK cells, resulting in a severely compromised innate immune response. Blocking NKG2A on CD8+ cells and NK cells in cancers modulated tumor growth, restoring CD8+ T and NK cell function. A recently proposed mechanism via which SARS-CoV-2 overrides innate immune response of the host is by over-expressing NKG2A on CD+ T and NK cells, culminating in functional exhaustion of the immune response against the viral pathogen. Monalizumab is an inhibiting antibody against NKG2A which can restore the function of CD8 + T and NK cells in cancers, successfully ceasing tumor progression with no significant side effects in Phase 2 clinical trials. We hypothesize that patients with severe COVID-19 have a severely compromised innate immune response and could be treated via the use of Monalizumab, interferon α, chloroquine, and other antiviral agents.

Title: Focus on Receptors for Coronaviruses with Special Reference to Angiotensin-converting Enzyme 2 as a Potential Drug Target - A Perspective. Content: Coronaviruses (CoVs) possess an enveloped, single, positive-stranded RNA genome which encodes for four membrane proteins, namely spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins 3-5 [1]. With regard to pathogenicity, S proteins are essential for viral entry into host cells [2, 3]. SARS-CoV binds to the angiotensin-converting enzyme (ACE)2 which is present on nonimmune cells, such as respiratory and intestinal epithelial cells, endothelial cells, kidney cells (renal tubules) and cerebral neurons and immune cells, such as alveolar monocytes/macrophages [4-6]. Of note, CD209L or liver/lymph node special intercellular adhesion molecule-3-grabbing non-integrin (SIGN) and dendritic cell (DC)-SIGN are alternative receptors for SARS-CoV but with lower affinity [7]. In the case of MERS-CoV, S proteins bind to the host cell receptor dipeptidyl peptidase 4 (DPP4 or CD26) which is broadly expressed on intestinal, alveolar, renal, hepatic and prostate cells as well as on activated leukocytes [8]. Then, viruses replicate in target cells with release of mature virions, which, in turn, invade new target cells [9]. Evidence has been provided that SARSCoV proteins are cleaved into two subunits, S1 and S2, respectively, and the amino acids 318-510 of the S1 represent the receptor-binding domain (RBD) which binds to ACE2 [10, 11]. Quite importantly, in the context of RBD there is the receptor-binding motif (RBM) (amino acids 424- 494), which accounts for complete binding to ACE2 [11]. Moreover, by means of two residues at positions 479 and 487 RBD allows virus progression and tropism [10, 11]. In the case of MERSCoV, its RBM binds to DPP4 with residues 484-567, thus, suggesting that its RBD differs from that of SARS-CoV [12, 13]. In a very recent paper, Wan and associates [14] have investigated the receptor recognition by COVID-19 (a new term to indicate the 2019-nCoV in Wuhan) on the bases of structural studies. In this respect, the sequence of COVID-19 RBM is similar to that of SARSCoV, thus, implicating that ACE2 may represent the binding receptors for COVID-19. Furthermore, gln493 residue of COVID-19 RBM seems to allow interaction with human ACE2, thus, suggesting the ability of this virus to infect human cells. According, to Wan and associates structural analysis [14], COVID-19 binds to human ACE2 with a lesser efficiency than human SARS-CoV (2002) but with higher affinity than human SARS-CoV (2003). Furthermore, same authors predicted that a single mutation at the 501 position may enhance the COVID-19 RBD binding capacity to human ACE2 and this evolution should be monitored in infected patients [14]. These predictive findings by Wan and associates [14] are confirmed by two contemporary studies by Letko and Muster [15] and Peng and associates [16]. In particular, the report by Peng and associates [16], points out the possible origin of COVID-19 from bats [16]. From a pathogenic point of view, evidence has been provided that binding of S2 to ACE2 receptor leads to its down-regulation with subsequent lung damage in the course of SARS-CoV infection [17]. Down-regulation of ACE2 causes excessive production of angiotensin (ANG) II by the related enzyme ACE with stimulation of ANG type 1a receptor (AT1R) and enhanced lung vascular permeability [18]. In particular, same authors have reported that recombinant ACE2 could attenuate severe acute lung injury in mice [18]. Moreover, Battle and associates [19] also proposed to use already available recombinant ACE2 for intercepting COVID-19 and attenuating infection. In the previous paragraphs, the presence of ACE2 on immune cells has been pointed out and, by analogy to epithelial cells, this receptor may also be down-regulated following viral entry. Therefore, in CoV-infected animal models and in infected humans further investigations are required to clarify a possible reduced expression of ACE2 on immune cells. In fact, in the course of SARS-CoV infection, a number of immune disorders have been detected. Three reports have demonstrated the ability of CoV to inhibit interferon (IFN)- production in the course of SARS acting as IFN antagonist [20-22]. In senescent Balb/c mice, depletion of T lymphocytes is associated to more severe interstitial pneumonitis and delayed clearance of SARS-CoV, thus, suggesting a protective role played by these cells [23]. In this connection, both SARS-CoV and MERS-CoV have been shown to induce T cell apoptosis, thus, aggravating the clinical course of disease [24, 25]. Quite interestingly, memory CD8+ T cells specific for SARS-CoV M and N proteins have been detected up to 11 years post-infection [26]. As far as humoral immune responsiveness is concerned, evidence has been provided that S1 subunit from MERS-CoV is highly immunogenic in mice [27]. Moreover, monoclonal antibodies have been shown to be highly neutralizing against MERS-CoV replication and endowed with post exposure effectiveness in susceptible mice [28, 29]. Human neutralizing antibodies have also been isolated from a recovered patient, thus, suggesting the role of humoral immunity in the control of the persistence of CoV in the host [30]. In particular, IgG response occurs early in infection and its prolonged production may serve for virus clearance during recovery also in view of the absence of viremia in convalescent sera from SARS patients [31]. According to current literature, severity of COVID-19 infection correlates with lymphopenia and patients who died from COVID-19 had lower lymphocyte counts when compared to survivors [32, 33]. These data suggest that lymphocyte-mediated anti-viral activity is poorly effective against COVID-19. Despite lymphopenia, evidence for an exaggerate release of proinflammatory cytokines [interleukin (IL)-1 and IL-6] has been reported in the course acute respiratory syndrome in COVID19 infected patients, thus, aggravating the clinical course of disease [34]. As recently reported, during COVID-19 pandemic in both Italy and China higher frequency of fatalities have been observed in the frail elderly population with previous comorbidities [35]. It is well known that decline of immunity occurs in ageing and, therefore, COVID-19 may gain easier access to the respiratory tract in frail elderly patients [36]. There is evidence that ACE2 protects from severe acute lung failure and operates as a negative regulator of the renin-angiotensin system (RAS) [18, 37]. It is well known that ANG II via activation of the AT1R promotes detrimental effects on the host, such as, vasoconstriction, reactive oxygen species generation, inflammation and matrix remodelling [38]. ACE2 counterbalances the noxious effects exhibited by ANG II and AT1R via activation of AT2R which arrests cell growth, inflammation and fibrosis [39]. In this framework, Gurwitz [40] proposed to use AT1R blockers, such as losartan, as a potential treatment of COVID-19 infection. In fact, losartan as well as olmesartan, used for treating hypertension in patients, were able to increase ACE2 expression after 28 days treatment of rats with myocardial infarction [41]. Then, Gurwitz suggests to evaluate severity of symptoms in COVID-19 infected patients under previous chronic treatment with AT1R blockers in comparison to COVID-19 infected patients who did not take AT1R blockers [40]. Quite interestingly, 75% of aged COVID-19 infected patients admitted to Italian hospitals had hypertension [unpublished data]. However, the putative effects of ACE-2 down-regulation on the cardiovascular system in the course of COVID-19 pandemic need more intensive studies. Taken together, these evidences suggest that CoV-induced down-regulation of ACE2 activates RAS with collateral damage to organs, such as lungs, in the course of SARS-related pneumonia. Then, putative therapeutic measures aimed at increasing ACE2 levels on respiratory epithelial cells should be taken into serious consideration. Quite interestingly, over the past few years, three key papers have demonstrated the ability of a polyphenol, resveratrol (RES), to experimentally deactivate the RAS system in maternal and post-weaning high fat diet, arterial ageing and high fat diet, respectively [42-44]. In all these experimental models, RES led to an increase of ACE2 with reduction of organ damage, such as liver steatosis and aorta media thickness and decrease of adipose tissue mass, respectively. As far as the mechanism of action of RES is concerned, this polyphenol is able to activate sirtuin (Sirt)1 [45-47]. In turn, Sirt1 down-regulates AT1R expression via ACE2 up-regulation [43, 48]. Of importance, Lin and associates [48] have demonstrated the ability of RES to in vitro inhibit MERS-CoV infection of Vero E6 cells, thus, prolonging cell survival in virtue of an anti-apoptotic mechanism. These findings suggest a direct antiviral effect exerted by RES. It would be very interestingly to evaluate the direct effects of RES on COVID-19, in vitro. The data above discussed strongly suggest, that RES, as an activators of ACE2, should be investigated in animal models of CoV-induced severe pneumonia, also taking into account the antioxidant, anti-inflammatory and immunomodulating effects exerted by polyphenols [49]. Then, successful animal studies may pave the way for RES-based human trials in COVID-infected patients. Note added in proof During the reviewing process of this perspective other related papers have been published. Hanff and associates [50] have discussed the possible association between COVID-19-associated cardiovascular mortality and dysregulation of the Renin Angiotensin System (RAS). From a pharmacologic point of view, RAS inhibition leads to upregulation of ACE2, thus, attenuating acute respiratory syndrome and myocarditis in COVID-19-infected patients. Conversely, increase in ACE2 expression may facilitate the access into the host of COVID-19, thus, aggravating the clinical picture. Such a dilemma would be solved by clinical trials based on RAS blo

Title: Beyond the Spike: identification of viral targets of the antibody response to SARS-CoV-2 in COVID-19 patients Content: Background: The SARS-CoV-2 virus emerged in December 2019 and caused a pandemic associated with a spectrum of COVID-19 disease ranging from asymptomatic to lethal infection. Serology testing is important for diagnosis of infection, determining infection attack rates and immunity in the population. It also informs vaccine development. Although several serology tests are in use, improving their specificity and sensitivity for early diagnosis on the one hand and for detecting past infection for population-based studies, are priorities. Methods: We evaluated the anti-SARS-CoV-2 antibody profiles to 15 SARS-CoV-2 antigens by cloning and expressing 15 open reading frames (ORFs) in mammalian cells and screened antibody responses to them in COVID-19 patients using the Luciferase Immunoprecipitation System (LIPS). Results: The LIPS technique allowed us to detect antibody responses in COVID-19 patients to 11 of the 15 SARS-CoV-2 antigens tested, identifying novel immunogenic targets. This technique shows that antigens ORF3b and ORF8 allow detection of antibody early in infection in a specific manner and reveals the immuno-dominance of the N antigen in COVID-19 patients. Conclusion: Our report provides an unbiased characterization of antibody responses to a range of SARS-CoV-2 antigens. The combination of 3 SARS-CoV-2 antibody LIPS assays, i.e. N, ORF3b, and ORF8, is sufficient to identify all COVID-19 patients of our cohort even at early time-points of illness, whilst Spike alone fails to do so. Furthermore, our study highlights the importance of investigating new immunogens NSP1, ORF3b, ORF7a and ORF8 which may mediate immune functions other than neutralization which may be beneficial or harmful to the patient.

Title: Pre-existing immunity against Ad vectors: Humoral, cellular, and innate response, what's important? Content: Pre-existing immunity against human adenovirus (HAd) serotype 5 derived vector in the human population is widespread, thus hampering its clinical use. Various components of the immune system, including neutralizing antibodies (nAbs), Ad specific T cells and type I IFN activated NK cells, contribute to dampening the efficacy of Ad vectors in individuals with pre-existing Ad immunity. In order to circumvent pre-existing immunity to adenovirus, numerous strategies, such as developing alternative Ad serotypes, varying immunization routes and utilizing prime-boost regimens, are under pre-clinical or clinical phases of development. However, these strategies mainly focus on one arm of pre-existing immunity. Selection of alternative serotypes has been largely driven by the absence in the human population of nAbs against them with little attention paid to cross-reactive Ad specific T cells. Conversely, varying the route of immunization appears to mainly rely on avoiding Ad specific tissue-resident T cells. Finally, prime-boost regimens do not actually circumvent pre-existing immunity but instead generate immune responses of sufficient magnitude to confer protection despite pre-existing immunity. Combining the above strategies and thus taking into account all components regulating pre-existing Ad immunity will help further improve the development of Ad vectors for animal and human use.

Title: Immune Response to Viruses: Antibody-Mediated Immunity Content: The notion about immunity to disease arose from the observation that those who recovered from an apparently contagious disease became resistant to a subsequent similar sickness. Much later it was shown that immunity is transferable by serum. The active serum components were identified to be immunoglobulins (Ig's), called antibodies. The enormous diversity of antibodies specific for distinct viruses, pathogens, and many other antigens is explained by clonal selection, whereby specific B-lymphocyte receptors recognize a particular antigen. The selected B-cells are triggered to undergo replication. A process of further cell differentiation and maturation ensues, leading to secretion of antibodies with high binding affinity toward the triggering antigen. Genes coding for the variable regions (involved in antigen binding) of Ig's are inherited as sets of gene fragments joined to form a complete gene in individual B-cells. This process and further hypermutations ensure the synthesis of diverse high affinity antibodies. The antibodies consist of pairs of light (L) and heavy (H) polypeptide chains. Variations in the constant portion of H-chains lead to production of Ig isotypes (IgM, IgA, IgD, IgE, and IgG (further subdivided into IgG1, IgG2, IgG3 and IgG4)), each having distinct effector functions. Host exposure to viruses leads to the production of antibodies with more than one specificity. Only some of these antibodies, recognizing so-called virus neutralization epitopes, diminish or eliminate virus infectivity. Other virus-specific antibodies play auxiliary roles or are ineffective. Sometimes antibodies cause enhancement of viral diseases or play a role in evasion of the immune system. Many antiviral immunoglobulins are being used for short-term pre-exposure prophylaxis or therapy. Long-term protective effects can be accomplished only by antibodies elicited by successful vaccination relying on the phenomenon of immunological memory. T-lymphocytes play a major role in initiating and maintaining immunity against subsequent virus exposure. Antibodies are one of the essential features of antigen triggered adaptive immunity. Initial early defense against viruses is provided by components of innate immunity which evolutionarily precedes adaptive immunity, and remains an essential part of defense against pathogens in humans.

Title: Harnessing the Power of T Cells: The Promising Hope for a Universal Influenza Vaccine Content: Next-generation vaccines that utilize T cells could potentially overcome the limitations of current influenza vaccines that rely on antibodies to provide narrow subtype-specific protection and are prone to antigenic mismatch with circulating strains. Evidence from animal models shows that T cells can provide heterosubtypic protection and are crucial for immune control of influenza virus infections. This has provided hope for the design of a universal vaccine able to prime against diverse influenza virus strains and subtypes. However, multiple hurdles exist for the realisation of a universal T cell vaccine. Overall primary concerns are: extrapolating human clinical studies, seeding durable effective T cell resident memory (Trm), population human leucocyte antigen (HLA) coverage, and the potential for T cell-mediated immune escape. Further comprehensive human clinical data is needed during natural infection to validate the protective role T cells play during infection in the absence of antibodies. Furthermore, fundamental questions still exist regarding the site, longevity and duration, quantity, and phenotype of T cells needed for optimal protection. Standardised experimental methods, and eventually simplified commercial assays, to assess peripheral influenza-specific T cell responses are needed for larger-scale clinical studies of T cells as a correlate of protection against influenza infection. The design and implementation of a T cell-inducing vaccine will require a consensus on the level of protection acceptable in the community, which may not provide sterilizing immunity but could protect the individual from severe disease, reduce the length of infection, and potentially reduce transmission in the community. Therefore, increasing the standard of care potentially offered by T cell vaccines should be considered in the context of pandemic preparedness and zoonotic infections, and in combination with improved antibody vaccine targeting methods. Current pandemic vaccine preparedness measures and ongoing clinical trials under-utilise T cell-inducing vaccines, reflecting the myriad questions that remain about how, when, where, and which T cells are needed to fight influenza virus infection. This review aims to bring together basic fundamentals of T cell biology with human clinical data, which need to be considered for the implementation of a universal vaccine against influenza that harnesses the power of T cells.

Title: Effects of an epitope-specific CD8+ T-cell response on murine coronavirus central nervous system disease: protection from virus replication and antigen spread and selection of epitope escape mutants. Content: Both CD4(+) and CD8(+) T cells are required for clearance of the murine coronavirus mouse hepatitis virus (MHV) during acute infection. We investigated the effects of an epitope-specific CD8(+) T-cell response on acute infection of MHV, strain A59, in the murine CNS. Mice with CD8(+) T cells specific for gp33-41 (an H-2D(b)-restricted CD8(+) T-cell epitope derived from lymphocytic choriomeningitis glycoprotein) were infected with a recombinant MHV-A59, also expressing gp33-41, as a fusion protein with enhanced green fluorescent protein (EGFP). By 5 days postinfection, these mice showed significantly (approximately 20-fold) lower titers of infectious virus in the brain compared to control mice. Furthermore mice with gp33-41-specific CD8(+) cells exhibited much reduced levels of viral antigen in the brain as measured by immunohistochemistry using an antibody directed against viral nucleocapsid. More than 90% of the viruses recovered from brain lysates of such protected mice, at 5 days postinfection, had lost the ability to express EGFP and had deletions in their genomes encompassing EGFP and gp33-41. In addition, genomes of viruses from about half the plaques that retained the EGFP gene had mutations within the gp33-41 epitope. On the other hand, gp33-41-specific cells failed to protect perforin-deficient mice from infection by the recombinant MHV expressing gp33, indicating that perforin-mediated mechanisms were needed. Virus recovered from perforin-deficient mice did not exhibit loss of EGFP expression and the gp33-41 epitope. These observations suggest that the cytotoxic T-cell response to gp33-41 exerts a strong immune pressure that quickly selects epitope escape mutants to gp33-41.

Title: Convergent Antibody Responses to SARS-CoV-2 Infection in Convalescent Individuals Content: During the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives. Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S). Although there is no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-2(1–5). Here we report on 149 COVID-19 convalescent individuals. Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal neutralizing titers ranging from undetectable in 33% to below 1:1000 in 79%, while only 1% showed titers >1:5000. Antibody cloning revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals. Despite low plasma titers, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (IC(50)s) as low as single digit ng/mL. Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

Title: Immune response to SARS-CoV-2 and mechanisms of immunopathological changes in COVID-19 Content: As a zoonotic disease that has already spread globally to several million human beings and possibly to domestic and wild animals, eradication of coronavirus disease 2019 (COVID-19) appears practically impossible. There is a pressing need to improve our understanding of the immunology of this disease to contain the pandemic by developing vaccines and medicines for the prevention and treatment of patients. In this review, we aim to improve our understanding on the immune response and immunopathological changes in patients linked to deteriorating clinical conditions such as cytokine storm, acute respiratory distress syndrome, autopsy findings and changes in acute-phase reactants, and serum biochemistry in COVID-19. Similar to many other viral infections, asymptomatic disease is present in a significant but currently unknown fraction of the affected individuals. In the majority of the patients, a 1-week, self-limiting viral respiratory disease typically occurs, which ends with the development of neutralizing antiviral T cell and antibody immunity. The IgM-, IgA-, and IgG-type virus-specific antibodies levels are important measurements to predict population immunity against this disease and whether cross-reactivity with other coronaviruses is taking place. High viral load during the first infection and repeated exposure to virus especially in healthcare workers can be an important factor for severity of disease. It should be noted that many aspects of severe patients are unique to COVID-19 and are rarely observed in other respiratory viral infections, such as severe lymphopenia and eosinopenia, extensive pneumonia and lung tissue damage, a cytokine storm leading to acute respiratory distress syndrome, and multiorgan failure. Lymphopenia causes a defect in antiviral and immune regulatory immunity. At the same time, a cytokine storm starts with extensive activation of cytokine-secreting cells with innate and adaptive immune mechanisms both of which contribute to a poor prognosis. Elevated levels of acute-phase reactants and lymphopenia are early predictors of high disease severity. Prevention of development to severe disease, cytokine storm, acute respiratory distress syndrome, and novel approaches to prevent their development will be main routes for future research areas. As we learn to live amidst the virus, understanding the immunology of the disease can assist in containing the pandemic and in developing vaccines and medicines to prevent and treat individual patients.

Title: Antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus Content: BACKGROUND: Public health risks associated to infection by human coronaviruses remain considerable and vaccination is a key option for preventing the resurgence of severe acute respiratory syndrome coronavirus (SARS-CoV). We have previously reported that antibodies elicited by a SARS-CoV vaccine candidate based on recombinant, full-length SARS-CoV Spike-protein trimers, trigger infection of immune cell lines. These observations prompted us to investigate the molecular mechanisms and responses to antibody-mediated infection in human macrophages. METHODS: We have used primary human immune cells to evaluate their susceptibility to infection by SARS-CoV in the presence of anti-Spike antibodies. Fluorescence microscopy and real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) were utilized to assess occurrence and consequences of infection. To gain insight into the underlying molecular mechanism, we performed mutational analysis with a series of truncated and chimeric constructs of fragment crystallizable γ receptors (FcγR), which bind antibody-coated pathogens. RESULTS: We show here that anti-Spike immune serum increased infection of human monocyte-derived macrophages by replication-competent SARS-CoV as well as Spike-pseudotyped lentiviral particles (SARS-CoVpp). Macrophages infected with SARS-CoV, however, did not support productive replication of the virus. Purified anti-viral IgGs, but not other soluble factor(s) from heat-inactivated mouse immune serum, were sufficient to enhance infection. Antibody-mediated infection was dependent on signaling-competent members of the human FcγRII family, which were shown to confer susceptibility to otherwise naïve ST486 cells, as binding of immune complexes to cell surface FcγRII was necessary but not sufficient to trigger antibody-dependent enhancement (ADE) of infection. Furthermore, only FcγRII with intact cytoplasmic signaling domains were competent to sustain ADE of SARS-CoVpp infection, thus providing additional information on the role of downstream signaling by FcγRII. CONCLUSIONS: These results demonstrate that human macrophages can be infected by SARS-CoV as a result of IgG-mediated ADE and indicate that this infection route requires signaling pathways activated downstream of binding to FcγRII receptors.

Title: T helper cell- and CD40-dependent germline IgM prevents chronic virus-induced demyelinating disease. Content: Generation of antiviral IgM is usually considered as a marker of a short-lived initial antibody response that is replaced by hypermutated and more-efficient IgG. However, once viruses have established a particular niche for their persistence (e.g., within the CNS), the immune system has to specifically mobilize a broad range of antimicrobial effectors to contain the pathogen in the long term. Infection of the CNS with the mouse hepatitis virus (MHV) provides a unique model situation in which the extent of inflammatory CNS disease is determined by the balance between antiviral immune control, viral replication, and immune-mediated damage. We show here that whereas antibody- or B cell-deficient mice failed to contain MHV CNS infection and developed progressive demyelinating disease, germline IgM produced in activation-induced cytidine deaminase-deficient mice (aicda(-/-)) provided long-term protection against the chronic multiple sclerosis-like disease. Furthermore, we found that appropriate B-cell activation within the CNS-draining lymph node and subsequent CXCR3-mediated migration of antiviral IgM-secreting cells to the infected CNS was dependent on CD40-mediated interaction of B cells with T helper cells. These data indicate that the CD40-mediated collaboration of T and B cells is critical to secure neuroprotective IgM responses during viral CNS infection.

Title: Type I Interferon Receptor Deficiency in Dendritic Cells Facilitates Systemic Murine Norovirus Persistence Despite Enhanced Adaptive Immunity Content: In order for a virus to persist, there must be a balance between viral replication and immune clearance. It is commonly believed that adaptive immunity drives clearance of viral infections and, thus, dysfunction or viral evasion of adaptive immunity is required for a virus to persist. Type I interferons (IFNs) play pleiotropic roles in the antiviral response, including through innate control of viral replication. Murine norovirus (MNoV) replicates in dendritic cells (DCs) and type I IFN signaling in DCs is important for early control of MNoV replication. We show here that the non-persistent MNoV strain CW3 persists systemically when CD11c positive DCs are unable to respond to type I IFN. Persistence in this setting is associated with increased early viral titers, maintenance of DC numbers, increased expression of DC activation markers and an increase in CD8 T cell and antibody responses. Furthermore, CD8 T cell function is maintained during the persistent phase of infection and adaptive immune cells from persistently infected mice are functional when transferred to Rag1 (-/-) recipients. Finally, increased early replication and persistence are also observed in mixed bone marrow chimeras where only half of the CD11c positive DCs are unable to respond to type I IFN. These findings demonstrate that increased early viral replication due to a cell-intrinsic innate immune deficiency is sufficient for persistence and a functional adaptive immune response is not sufficient for viral clearance.

Title: B-cell responses in patients who have recovered from severe acute respiratory syndrome target a dominant site in the S2 domain of the surface spike glycoprotein. Content: Severe acute respiratory syndrome (SARS) is a recently emerged infectious disease caused by a novel strain of coronavirus. Examination of the immune responses of patients who have recovered from SARS should provide important information for design of a safe and effective vaccine. We determined the continuous viral epitopes targeted by antibodies in plasma samples from convalescent SARS patients through biopanning with a vast M13 phage display dodecapeptide library. These epitopes converged to very short peptide fragments, one on each of the structural proteins spike and nucleocapsid and the nonstructural proteins 3a, 9b, and nsp 3. Immunoassays found that most of the patients who had recovered from SARS developed complementary antibodies to the epitope-rich region on the spike S2 protein, indicating that this is an immunodominant site on the viral envelope comprising the spike, matrix, and small envelope glycoproteins. These S2-targeting antibodies were shown to effectively neutralize the coronavirus, indicating that they provided protective immunity to help the patients recover from the viral infection. These results suggest that the SARS coronavirus might have an antigenic profile distinct from those of other human or animal coronaviruses. Due to the tested safety and protective effects of the convalescent-phase serological antibodies, identification of their complementary antigens may enable the design of an epitope-based vaccine to prevent potential antibody-mediated immunopathology.

Title: CD8(+) T cells in HIV control, cure and prevention Content: HIV infection can be effectively treated by lifelong administration of combination antiretroviral therapy, but an effective vaccine will likely be required to end the HIV epidemic. Although the majority of current vaccine strategies focus on the induction of neutralizing antibodies, there is substantial evidence that cellular immunity mediated by CD8(+) T cells can sustain long-term disease-free and transmission-free HIV control and may be harnessed to induce both therapeutic and preventive antiviral effects. In this Review, we discuss the increasing evidence derived from individuals who spontaneously control infection without antiretroviral therapy as well as preclinical immunization studies that provide a clear rationale for renewed efforts to develop a CD8(+) T cell-based HIV vaccine in conjunction with B cell vaccine efforts. Further, we outline the remaining challenges in translating these findings into viable HIV prevention, treatment and cure strategies.

Title: Is copper beneficial for COVID-19 patients? Content: Copper (Cu) is an essential micronutrient for both pathogens and the hosts during viral infection. Cu is involved in the functions of critical immune cells such as T helper cells, B cells, neutrophils natural killer (NK) cells, and macrophages. These blood cells are involved in the killing of infectious microbes, in cell-mediated immunity and the production of specific antibodies against the pathogens. Cu-deficient humans show an exceptional susceptibility to infections due to the decreased number and function of these blood cells. Besides, Cu can kill several infectious viruses such as bronchitis virus, poliovirus, human immunodeficiency virus type 1(HIV-1), other enveloped or nonenveloped, single- or double-stranded DNA and RNA viruses. Moreover, Cu has the potent capacity of contact killing of several viruses, including SARS-CoV-2. Since the current outbreak of the COVID-19 continues to develop, and there is no vaccine or drugs are currently available, the critical option is now to make the immune system competent to fight against the SARS-CoV-2. Based on available data, we hypothesize that enrichment of plasma copper levels will boost both the innate and adaptive immunity in people. Moreover, owing to its potent antiviral activities, Cu may also act as a preventive and therapeutic regime against COVID-19.

Title: Is Copper beneficial for COVID-19 patients? Content: Abstract Copper (Cu) is an essential micronutrient for both pathogens and the hosts during viral infection. Cu is involved in the functions of critical immune cells such as T helper cells, B cells, neutrophils natural killer (NK) cells, and macrophages. These blood cells are involved in the killing of infectious microbes, in cell-mediated immunity and the production of specific antibodies against the pathogens. Cu-deficient humans show an exceptional susceptibility to infections due to the decreased number and function of these blood cells. Besides, Cu can kill several infectious viruses such as bronchitis virus, poliovirus, human immunodeficiency virus type 1(HIV-1), other enveloped or nonenveloped, single- or double-stranded DNA and RNA viruses. Moreover, Cu has the potent capacity of contact killing of several viruses, including SARS‐CoV‐2. Since the current outbreak of the COVID-19 continues to develop, and there is no vaccine or drugs are currently available, the critical option is now to make the immune system competent to fight against the SARS‐CoV‐2. Based on available data, we hypothesize that enrichment of plasma copper levels will boost both the innate and adaptive immunity in people. Moreover, owing to its potent antiviral activities, Cu may also act as a preventive and therapeutic regime against COVID-19.

Title: Convergent antibody responses to SARS-CoV-2 in convalescent individuals Content: During the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives. Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S). Although there is no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21-5. Here we report on 149 COVID-19 convalescent individuals. Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titres: less than 1:50 in 33% and below 1:1,000 in 79%, while only 1% showed titres above 1:5,000. Antibody sequencing revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals. Despite low plasma titres, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (IC50 values) as low as single digit nanograms per millitre. Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

Title: Convergent antibody responses to SARS-CoV-2 in convalescent individuals. Content: During the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives. Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S). Although there is no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21-5. Here we report on 149 COVID-19 convalescent individuals. Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titres: less than 1:50 in 33% and below 1:1,000 in 79%, while only 1% showed titres above 1:5,000. Antibody sequencing revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals. Despite low plasma titres, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (IC50 values) as low as single digit nanograms per millitre. Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

Title: Focus on Receptors for Coronaviruses with Special Reference to Angiotensin-converting Enzyme 2 as a Potential Drug Target - A Perspective Content: Coronaviruses (CoVs) possess an enveloped, single, positive-stranded RNA genome which encodes for four membrane proteins, namely spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins 3-5 [1]. With regard to pathogenicity, S proteins are essential for viral entry into host cells [2, 3]. SARS-CoV binds to the angiotensin-converting enzyme (ACE)2 which is present on nonimmune cells, such as respiratory and intestinal epithelial cells, endothelial cells, kidney cells (renal tubules) and cerebral neurons and immune cells, such as alveolar monocytes/macrophages [4-6]. Of note, CD209L or liver/lymph node special intercellular adhesion molecule-3-grabbing non-integrin (SIGN) and dendritic cell (DC)-SIGN are alternative receptors for SARS-CoV but with lower affinity [7]. In the case of MERS-CoV, S proteins bind to the host cell receptor dipeptidyl peptidase 4 (DPP4 or CD26) which is broadly expressed on intestinal, alveolar, renal, hepatic and prostate cells as well as on activated leukocytes [8]. Then, viruses replicate in target cells with release of mature virions, which, in turn, invade new target cells [9]. Evidence has been provided that SARSCoV proteins are cleaved into two subunits, S1 and S2, respectively, and the amino acids 318-510 of the S1 represent the receptor-binding domain (RBD) which binds to ACE2 [10, 11]. Quite importantly, in the context of RBD there is the receptor-binding motif (RBM) (amino acids 424- 494), which accounts for complete binding to ACE2 [11]. Moreover, by means of two residues at positions 479 and 487 RBD allows virus progression and tropism [10, 11]. In the case of MERSCoV, its RBM binds to DPP4 with residues 484-567, thus, suggesting that its RBD differs from that of SARS-CoV [12, 13]. In a very recent paper, Wan and associates [14] have investigated the receptor recognition by COVID-19 (a new term to indicate the 2019-nCoV in Wuhan) on the bases of structural studies. In this respect, the sequence of COVID-19 RBM is similar to that of SARSCoV, thus, implicating that ACE2 may represent the binding receptors for COVID-19. Furthermore, gln493 residue of COVID-19 RBM seems to allow interaction with human ACE2, thus, suggesting the ability of this virus to infect human cells. According, to Wan and associates structural analysis [14], COVID-19 binds to human ACE2 with a lesser efficiency than human SARS-CoV (2002) but with higher affinity than human SARS-CoV (2003). Furthermore, same authors predicted that a single mutation at the 501 position may enhance the COVID-19 RBD binding capacity to human ACE2 and this evolution should be monitored in infected patients [14]. These predictive findings by Wan and associates [14] are confirmed by two contemporary studies by Letko and Muster [15] and Peng and associates [16]. In particular, the report by Peng and associates [16], points out the possible origin of COVID-19 from bats [16]. From a pathogenic point of view, evidence has been provided that binding of S2 to ACE2 receptor leads to its down-regulation with subsequent lung damage in the course of SARS-CoV infection [17]. Down-regulation of ACE2 causes excessive production of angiotensin (ANG) II by the related enzyme ACE with stimulation of ANG type 1a receptor (AT1R) and enhanced lung vascular permeability [18]. In particular, same authors have reported that recombinant ACE2 could attenuate severe acute lung injury in mice [18]. Moreover, Battle and associates [19] also proposed to use already available recombinant ACE2 for intercepting COVID-19 and attenuating infection. In the previous paragraphs, the presence of ACE2 on immune cells has been pointed out and, by analogy to epithelial cells, this receptor may also be down-regulated following viral entry. Therefore, in CoV-infected animal models and in infected humans further investigations are required to clarify a possible reduced expression of ACE2 on immune cells. In fact, in the course of SARS-CoV infection, a number of immune disorders have been detected. Three reports have demonstrated the ability of CoV to inhibit interferon (IFN)-ï¢ production in the course of SARS acting as IFN antagonist [20-22]. In senescent Balb/c mice, depletion of T lymphocytes is associated to more severe interstitial pneumonitis and delayed clearance of SARS-CoV, thus, suggesting a protective role played by these cells [23]. In this connection, both SARS-CoV and MERS-CoV have been shown to induce T cell apoptosis, thus, aggravating the clinical course of disease [24, 25]. Quite interestingly, memory CD8+ T cells specific for SARS-CoV M and N proteins have been detected up to 11 years post-infection [26]. As far as humoral immune responsiveness is concerned, evidence has been provided that S1 subunit from MERS-CoV is highly immunogenic in mice [27]. Moreover, monoclonal antibodies have been shown to be highly neutralizing against MERS-CoV replication and endowed with post exposure effectiveness in susceptible mice [28, 29]. Human neutralizing antibodies have also been isolated from a recovered patient, thus, suggesting the role of humoral immunity in the control of the persistence of CoV in the host [30]. In particular, IgG response occurs early in infection and its prolonged production may serve for virus clearance during recovery also in view of the absence of viremia in convalescent sera from SARS patients [31]. According to current literature, severity of COVID-19 infection correlates with lymphopenia and patients who died from COVID-19 had lower lymphocyte counts when compared to survivors [32, 33]. These data suggest that lymphocyte-mediated anti-viral activity is poorly effective against COVID-19. Despite lymphopenia, evidence for an exaggerate release of proinflammatory cytokines [interleukin (IL)-1 and IL-6] has been reported in the course acute respiratory syndrome in COVID19 infected patients, thus, aggravating the clinical course of disease [34]. As recently reported, during COVID-19 pandemic in both Italy and China higher frequency of fatalities have been observed in the frail elderly population with previous comorbidities [35]. It is well known that decline of immunity occurs in ageing and, therefore, COVID-19 may gain easier access to the respiratory tract in frail elderly patients [36]. There is evidence that ACE2 protects from severe acute lung failure and operates as a negative regulator of the renin-angiotensin system (RAS) [18, 37]. It is well known that ANG II via activation of the AT1R promotes detrimental effects on the host, such as, vasoconstriction, reactive oxygen species generation, inflammation and matrix remodelling [38]. ACE2 counterbalances the noxious effects exhibited by ANG II and AT1R via activation of AT2R which arrests cell growth, inflammation and fibrosis [39]. In this framework, Gurwitz [40] proposed to use AT1R blockers, such as losartan, as a potential treatment of COVID-19 infection. In fact, losartan as well as olmesartan, used for treating hypertension in patients, were able to increase ACE2 expression after 28 days treatment of rats with myocardial infarction [41]. Then, Gurwitz suggests to evaluate severity of symptoms in COVID-19 infected patients under previous chronic treatment with AT1R blockers in comparison to COVID-19 infected patients who did not take AT1R blockers [40]. Quite interestingly, 75% of aged COVID-19 infected patients admitted to Italian hospitals had hypertension [unpublished data]. However, the putative effects of ACE-2 down-regulation on the cardiovascular system in the course of COVID-19 pandemic need more intensive studies. Taken together, these evidences suggest that CoV-induced down-regulation of ACE2 activates RAS with collateral damage to organs, such as lungs, in the course of SARS-related pneumonia. Then, putative therapeutic measures aimed at increasing ACE2 levels on respiratory epithelial cells should be taken into serious consideration. Quite interestingly, over the past few years, three key papers have demonstrated the ability of a polyphenol, resveratrol (RES), to experimentally deactivate the RAS system in maternal and post-weaning high fat diet, arterial ageing and high fat diet, respectively [42-44]. In all these experimental models, RES led to an increase of ACE2 with reduction of organ damage, such as liver steatosis and aorta media thickness and decrease of adipose tissue mass, respectively. As far as the mechanism of action of RES is concerned, this polyphenol is able to activate sirtuin (Sirt)1 [45-47]. In turn, Sirt1 down-regulates AT1R expression via ACE2 up-regulation [43, 48]. Of importance, Lin and associates [48] have demonstrated the ability of RES to in vitro inhibit MERS-CoV infection of Vero E6 cells, thus, prolonging cell survival in virtue of an anti-apoptotic mechanism. These findings suggest a direct antiviral effect exerted by RES. It would be very interestingly to evaluate the direct effects of RES on COVID-19, in vitro. The data above discussed strongly suggest, that RES, as an activators of ACE2, should be investigated in animal models of CoV-induced severe pneumonia, also taking into account the antioxidant, anti-inflammatory and immunomodulating effects exerted by polyphenols [49]. Then, successful animal studies may pave the way for RES-based human trials in COVID-infected patients. Note added in proof During the reviewing process of this perspective other related papers have been published. Hanff and associates [50] have discussed the possible association between COVID-19-associated cardiovascular mortality and dysregulation of the Renin Angiotensin System (RAS). From a pharmacologic point of view, RAS inhibition leads to upregulation of ACE2, thus, attenuating acute respiratory syndrome and myocarditis in COVID-19-infected patients. Conversely, increase in ACE2 expression may facilitate the access into the host of COVID-19, thus, aggravating the clinical picture. Such a dilemma would be solved byclinical trials based on RAS blockade or initiation and monitoring related effects. Contemporarily, Danser and associates [51] claim that there is no evidence to stop RAS blockers in the course of COVID-19 infection. In fact, there are no available data which support that ACE inhibitors or ANG II type I receptor blockers increase COVID-19 infection via its binding to ACE2. Finally, Kuster and associates [52] write that there are no data on the strict relationship between ACE2 activity and SARS-CoV2 mortality. Moreover, in the SARSCoV2, cells expressing ACE2 were not attacked by the virus, while cells lacking ACE2 were bound by the SARS-CoV2 virus [53]. These findings suggest that also in the case of RES effects on COVID-19 infection, the dual role of ACE2 should be taken into serious consideration.

Title: Control of HPV-associated tumors by innovative therapeutic HPV DNA vaccine in the absence of CD4+ T cells Content: Human papillomavirus (HPV) infections are particularly problematic for HIV + and solid organ transplant patients with compromised CD4+ T cell-dependent immunity as they produce more severe and progressive disease compared to healthy individuals. There are no specific treatments for chronic HPV infection, resulting in an urgent unmet need for a modality that is safe and effective for both immunocompromised and otherwise normal patients with recalcitrant disease. DNA vaccination is attractive because it avoids the risks of administration of live vectors to immunocompromised patients, and can induce potent HPV-specific cytotoxic T cell responses. We have developed a DNA vaccine (pNGVL4a-hCRTE6E7L2) encoding calreticulin (CRT) fused to E6, E7 and L2 proteins of HPV-16, the genotype associated with approximately 90% vaginal, vulvar, anal, penile and oropharyngeal HPV-associated cancers and the majority of cervical cancers. Administration of the DNA vaccine by intramuscular (IM) injection followed by electroporation induced significantly greater HPV-specific immune responses compared to IM injection alone or mixed with alum. Furthermore, pNGVL4a-hCRTE6E7L2 DNA vaccination via electroporation of mice carrying an intravaginal HPV-16 E6/E7-expressing syngeneic tumor demonstrated more potent therapeutic effects than IM vaccination alone. Of note, administration of the DNA vaccine by IM injection followed by electroporation elicited potent E6 and E7-specific CD8+ T cell responses and antitumor effects despite CD4+ T cell-depletion, although no antibody response was detected. While CD4+ T cell-depletion did reduce the E6 and E7-specific CD8+ T cell response, it remained sufficient to prevent subcutaneous tumor growth and to eliminate circulating tumor cells in a model of metastatic HPV-16+ cancer. Thus, the antibody response was CD4-dependent, whereas CD4+ T cell help enhanced the E6/E7-specific CD8+ T cell immunity, but was not required. Taken together, our data suggest that pNGVL4a-hCRTE6E7L2 DNA vaccination via electroporation warrants testing in otherwise healthy patients and those with compromised CD4+ T cell immunity to treat HPV-16-associated anogenital disease and cancer.

Title: Broad phenotypic alterations and potential dysfunctions of lymphocytes in COVID-19 recovered individuals Content: Background Lymphopenia is a typical symptom in the COVID-19 patients. While millions of patients are clinical recovered, little is known about the immune status of lymphocytes in these individuals. Methods A clinical recovered cohort (CR) of 55 COVID-19 individuals (discharged from hospital 4 to 11 weeks), and 55 age and sex matched healthy donors cohort (HD) were recruited. Detailed analysis on phenotype of the lymphocytes in peripheral blood mononuclear cells (PBMCs) was performed by flow cytometry. Findings Compared with cohort HD, the CD8+ T cells in cohort CR had higher Teff and Tem, but lower Tc1 (IFN-{gamma}+), Tc2 (IL-4+) and Tc17 (IL-17A+) frequencies. The CD4+ T cells of CR had decreased frequency, especially on the Tcm subset. Moreover, CD4+ T cells of CR expressed lower PD-1 and had lower frequencies of Th1 (IFN-{gamma}+), Th2 (IL-4+), Th17 (IL-17A+) as well as circulating Tfh (CXCR5+PD-1+). Accordingly, isotype-switched memory B cell (IgM-CD20hi) in CR had significantly lower proportion in B cells, though level of activation marker CD71 elevated. For CD3-HLA-DRlo lymphocytes of CR, besides levels of IFN-{gamma}, Granzyme B and T-bet were lower, the correlation between T-bet and IFN-{gamma} became irrelevant. In addition, taken into account of discharged days, all the lowered function associated phenotypes showed no recovery tendency within whole observation period. Interpretation The CR COVID-19 individuals still showed remarkable phenotypic alterations in lymphocytes after clinical recovery 4 to 11 weeks. This suggests SARS-CoV-2 infection imprints profoundly on lymphocytes and results in long-lasting potential dysfunctions.

Title: Immunogenic profile of SARS-CoV-2 spike in individuals recovered from COVID-19 Content: The rapid global spread of SARS-CoV-2 and resultant mortality and social disruption have highlighted the need to better understand coronavirus immunity to expedite vaccine development efforts. Multiple candidate vaccines, designed to elicit protective neutralising antibodies targeting the viral spike glycoprotein, are rapidly advancing to clinical trial. However, the immunogenic properties of the spike protein in humans are unresolved. To address this, we undertook an in-depth characterisation of humoral and cellular immunity against SARS-CoV-2 spike in humans following mild to moderate SARS-CoV-2 infection. We find serological antibody responses against spike are routinely elicited by infection and correlate with plasma neutralising activity and capacity to block ACE2/RBD interaction. Expanded populations of spike-specific memory B cells and circulating T follicular helper cells (cTFH) were detected within convalescent donors, while responses to the receptor binding domain (RBD) constitute a minor fraction. Using regression analysis, we find high plasma neutralisation activity was associated with increased spike-specific antibody, but notably also with the relative distribution of spike-specific cTFH subsets. Thus both qualitative and quantitative features of B and T cell immunity to spike constitute informative biomarkers of the protective potential of novel SARS-CoV-2 vaccines.

Title: Response of memory CD8+ T cells to severe acute respiratory syndrome (SARS) coronavirus in recovered SARS patients and healthy individuals. Content: To date, the pathogenesis of severe acute respiratory syndrome (SARS) in humans is still not well understood. SARS coronavirus (SARS-CoV)-specific CTL responses, in particular their magnitude and duration of postinfection immunity, have not been extensively studied. In this study, we found that heat-inactivated SARS-CoV elicited recall CTL responses to newly identified spike protein-derived epitopes (SSp-1, S978, and S1202) in peripheral blood of all HLA-A*0201(+) recovered SARS patients over 1 year postinfection. Intriguingly, heat-inactivated SARS-CoV elicited recall-like CTL responses to SSp-1 but not to S978, S1202, or dominant epitopes from several other human viruses in 5 of 36 (13.8%) HLA-A*0201(+) healthy donors without any contact history with SARS-CoV. SSp-1-specific CTLs expanded from memory T cells of both recovered SARS patients, and the five exceptional healthy donors shared a differentiated effector CTL phenotype, CD45RA(+)CCR7(-)CD62L(-), and expressed CCR5 and CD44. However, compared with the high avidity of SSp-1-specific CTLs derived from memory T cells of recovered SARS patients, SSp-1-specific CTLs from the five exceptional healthy donors were of low avidity, as determined by their rapid tetramer dissociation kinetics and reduced cytotoxic reactivity, IFN-gamma secretion, and intracellular production of IFN-gamma, TNF-alpha, perforin, and granzyme A. These results indicate that SARS-CoV infection induces strong and long-lasting CTL-mediated immunity in surviving SARS patients, and that cross-reactive memory T cells to SARS-CoV may exist in the T cell repertoire of a small subset of healthy individuals and can be reactivated by SARS-CoV infection.

Title: Upregulation of CD47 Is a Host Checkpoint Response to Pathogen Recognition Content: It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 "don't eat me" signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions. A CD47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but CD47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that encode no mimic. CD47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (PRRs). Furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis C patients, upregulated CD47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. Indeed, results from antibody-mediated CD47 blockade experiments as well as CD47 knockout mice revealed an immunosuppressive role for CD47 during infections with lymphocytic choriomeningitis virus and Mycobacterium tuberculosis Since CD47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify CD47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents.IMPORTANCE Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile.

Title: SARS-CoV-2 epitopes are recognized by a public and diverse repertoire of human T-cell receptors Content: Understanding the determinants of adaptive immune response to SARS-CoV-2 is critical for fighting the ongoing COVID-19 pandemic. Here we assayed both antibody and T-cell reactivity to SARS-CoV-2 antigens in COVID-19 convalescent patients and healthy donors sampled before and during the pandemic. Our results show that while anti-SARS-CoV-2 antibodies can distinguish convalescent patients from healthy donors, the magnitude of T-cell response was more pronounced in healthy donors sampled during COVID-19 pandemic than in donors sampled before the outbreak. This hints at the possibility that some individuals have encountered the virus but were protected by T-cell cross-reactivity observed. A public and diverse T-cell response was observed for two A*02-restricted SARS-CoV-2 epitopes, revealing a set of T-cell receptor motifs displaying germline-encoded features. Bulk CD4+ and CD8+ T-cell response to SARS-CoV-2 glycoprotein S is characterized by multiple groups of homologous T-cell receptor sequences some of which are shared across multiple donors, indicating the existence of immunodominant epitopes. Overall, our findings indicate that T cells form an efficient response to SARS-CoV-2 and alongside the antibodies can serve as a useful biomarker for surveying SARS-CoV-2 exposure and immunity. We hope that data, including the set of specific T-cell receptors identified in this study can serve as a basis for future developments of SARS-CoV-2 vaccinations and monitoring.

Title: Prevention of virus persistence and protection against immunopathology after Borna disease virus infection of the brain by a novel Orf virus recombinant. Content: The Parapoxvirus Orf virus represents a promising candidate for novel vector vaccines due to its immune modulating properties even in nonpermissive hosts such as mouse or rat. The highly attenuated Orf virus strain D1701 was used to generate a recombinant virus (D1701-VrVp40) expressing nucleoprotein p40 of Borna disease virus, which represents a major antigen for the induction of a Borna disease virus-specific humoral and cellular immune response. Infection with Borna disease virus leads to distinct neurological symptoms mediated by the invasion of activated specific CD8+ T cells into the infected brain. Usually, Borna disease virus is not cleared from the brain but rather persists in neural cells. In the present study we show for the first time that intramuscular application of the D1701-VrVp40 recombinant protected rats against Borna disease, and importantly, virus clearance from the infected brain was demonstrated in immunized animals. Even 4 and 8 months after the last immunization, all immunized animals were still protected against the disease. Initial characterization of the immune cells attracted to the infected brain areas suggested that D1701-VrVp40 mediated induction of B cells and antibody-producing plasma cells as well as T cells. These findings suggest the induction of various defense mechanisms against Borna disease virus. First studies on the role of antiviral cytokines indicated that D1701-VrVp40 immunization did not lead to an enhanced early response of gamma or alpha interferon or tumor necrosis factor alpha. Collectively, this study describes the potential of the Orf virus vector system in mediating long-lasting, protective antiviral immunity and eliminating this persistent virus infection without provoking massive neuronal damage.

Title: Reflection on passive immunotherapy in those who need most: some novel strategic arguments for obtaining safer therapeutic plasma or autologous antibodies from recovered COVID ‐19 infected patients Content: COVID‐19 pandemic is an emerging new human disease, where no vaccines, or monoclonal antibodies (mAbs), or drugs are currently available for therapy. Active vaccination requires the induction of an immune response against a given agent to a susceptible individual for the purpose of preventing or treating an infectious disease and this usually takes time to develop. Thus, the use of existing autologous Ab administration, obtainable from recovered COVID 19 patients, after 2 weeks recovery, is the best and the most practical strategy for providing immediate passive immunity to susceptible recipients in need.

Title: T-cell immune responses in the brain and their relevance for cerebral malignancies. Content: In order that cellular immune responses afford protection without risk to sensitive normal tissue, they must be adapted to individual tissues of the body. Nowhere is this more critical than for the brain, where various passive and active mechanisms maintain a state of immune privilege that can limit high magnitude immune responses. Nevertheless, it is now clear that immune responses are induced to antigens in the brain, including those expressed by cerebral malignancies. We discuss hypotheses of how this can occur, although details such as which antigen presenting cells are involved remain to be clarified. Antitumor responses induced spontaneously are insufficient to eradicate malignant astrocytomas; many studies suggest that this can be explained by a combination of low level immune response induction and tumor mediated immunosuppression. A clinical objective currently pursued is to use immunotherapy to ameliorate antitumour immunity. This will necessitate a high level immune response to ensure sufficient effector cells reach the tumor bed, focused cytotoxicity to eradicate malignant cells with little collateral damage to critical normal cells, and minimal inflammation. To achieve these aims, priority should be given to identifying more target antigens in astrocytoma and defining those cells present in the brain parenchyma that are essential to maintain antitumour effector function without exacerbating inflammation. If we are armed with better understanding of immune interactions with brain tumor cells, we can realistically envisage that immunotherapy will one day offer hope to patients with currently untreatable neoplastic diseases of the CNS.

Title: Severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus. Content: Vaccines against infectious bronchitis of chickens (Gallus gallus domesticus) have arguably been the most successful, and certainly the most widely used, of vaccines for diseases caused by coronaviruses, the others being against bovine, canine, feline and porcine coronaviruses. Infectious bronchitis virus (IBV), together with the genetically related coronaviruses of turkey (Meleagris gallopovo) and ring-necked pheasant (Phasianus colchicus), is a group 3 coronavirus, severe acute respiratory syndrome (SARS) coronavirus being tentatively in group 4, the other known mammalian coronaviruses being in groups 1 and 2. IBV replicates not only in respiratory tissues (including the nose, trachea, lungs and airsacs, causing respiratory disease), but also in the kidney (associated with minor or major nephritis), oviduct, and in many parts of the alimentary tract--the oesophagus, proventriculus, duodenum, jejunum, bursa of Fabricius, caecal tonsils (near the distal end of the tract), rectum and cloaca (the common opening for release of eggs and faeces), usually without clinical effects. The virus can persist, being re-excreted at the onset of egg laying (4 to 5 months of age), believed to be a consequence of the stress of coming into lay. Genetic lines of chickens differ in the extent to which IBV causes mortality in chicks, and in respect of clearance of the virus after the acute phase. Live attenuated (by passage in chicken embryonated eggs) IBV strains were introduced as vaccines in the 1950s, followed a couple of decades later by inactivated vaccines for boosting protection in egg-laying birds. Live vaccines are usually applied to meat-type chickens at 1 day of age. In experimental situations this can result in sterile immunity when challenged by virulent homologous virus. Although 100% of chickens may be protected (against clinical signs and loss of ciliary activity in trachea), sometimes 10% of vaccinated chicks do not respond with a protective immune response. Protection is short lived, the start of the decline being apparent 9 weeks after vaccination with vaccines based on highly attenuated strains. IBV exists as scores of serotypes (defined by the neutralization test), cross-protection often being poor. Consequently, chickens may be re-vaccinated, with the same or another serotype, two or three weeks later. Single applications of inactivated virus has generally led to protection of <50% of chickens. Two applications have led to 90 to 100% protection in some reports, but remaining below 50% in others. In practice in the field, inactivated vaccines are used in laying birds that have previously been primed with two or three live attenuated virus vaccinations. This increases protection of the laying birds against egg production losses and induces a sustained level of serum antibody, which is passed to progeny. The large spike glycoprotein (S) comprises a carboxy-terminal S2 subunit (approximately 625 amino acid residues), which anchors S in the virus envelope, and an amino-terminal S1 subunit (approximately 520 residues), believed to largely form the distal bulbous part of S. The S1 subunit (purified from IBV virus, expressed using baculovirus or expressed in birds from a fowlpoxvirus vector) induced virus neutralizing antibody. Although protective immune responses were induced, multiple inoculations were required and the percentage of protected chickens was too low (<50%) for commercial application. Remarkably, expression of S1 in birds using a non-pathogenic fowl adenovirus vector induced protection in 90% and 100% of chickens in two experiments. Differences of as little as 5% between the S1 sequences can result in poor cross-protection. Differences in S1 of 2 to 3% (10 to 15 amino acids) can change serotype, suggesting that a small number of epitopes are immunodominant with respect to neutralizing antibody. Initial studies of the role of the IBV nucleocapsid protein (N) in immunity suggested that immunization with bacterially expressed N, while not inducing protection directly, improved the induction of protection by a subsequent inoculation with inactivated IBV. In another study, two intramuscular immunizations of a plasmid expressing N induced protective immunity. The basis of immunity to IBV is not well understood. Serum antibody levels do not correlate with protection, although local antibody is believed to play a role. Adoptive transfer of IBV-infection-induced alphabeta T cells bearing CD8 antigen protected chicks from challenge infection. In conclusion, live attenuated IBV vaccines induce good, although short-lived, protection against homologous challenge, although a minority of individuals may respond poorly. Inactivated IBV vaccines are insufficiently efficacious when applied only once and in the absence of priming by live vaccine. Two applications of inactivated IBV are much more efficacious, although this is not a commercially viable proposition in the poultry industry. However, the cost and logistics of multiple application of a SARS inactivated vaccine would be more acceptable for the protection of human populations, especially if limited to targeted groups (e.g. health care workers and high-risk contacts). Application of a SARS vaccine is perhaps best limited to a minimal number of targeted individuals who can be monitored, as some vaccinated persons might, if infected by SARS coronavirus, become asymptomatic excretors of virus, thereby posing a risk to non-vaccinated people. Looking further into the future, the high efficacy of the fowl adenovirus vector expressing the IBV S1 subunit provides optimism for a live SARS vaccine, if that were deemed to be necessary, with the possibility of including the N protein gene.

Title: Recovery from Infection Content: This chapter discusses the immunological factors associated in the recovery of host from infections. Antibody, T cells, natural killer cells, complement, phagocytes, and interferon are involved in the response to nearly all infections and, without any doubt, are together responsible for recovery. There is good evidence that T-cell-mediated immunity is of supreme importance in recovery from a variety of microbial infections. These are the infections in which the microorganism replicates intracellularly. Tissue responses in the host bear the hallmarks of T-cell involvement, the infiltrating cells consisting primarily of lymphocytes and macrophages. Inflammation, whether induced by immunological reactions, tissue damage or microbial products, plays a vital role in recovery from infection. Inflammation is necessary for the proper functioning of the immune defenses because it focuses all circulating antimicrobial factors onto the site of infection. Complement can carry out antimicrobial activities. However, there is little direct evidence that the antimicrobial activities of complement are in fact important in the body. Interferons also act on uninfected cells, binding to a cell surface receptor and activating a number of genes involved in immunity to viruses. Multimechanistic recovery is illustrated in the chapter and other issues, such as the role of temperature, tissue repair, and resistance to re-infection are discussed.

Title: Suppression of innate immune pathology by regulatory T cells during Influenza A virus infection of immunodeficient mice. Content: The viral infection of higher vertebrates elicits potent innate and adaptive host immunity. However, an excessive or inappropriate immune response also may lead to host pathology that often is more severe than the direct effects of viral replication. Therefore, several mechanisms exist that regulate the magnitude and class of the immune response. Here, we have examined the potential involvement of regulatory T (Treg) cells in limiting pathology induced by influenza A virus (IAV) infection. Using lymphocyte-deficient mice as hosts, we showed that Treg cell reconstitution resulted in a significant delay in weight loss and prolonged survival following infection. The adoptively transferred Treg cells did not affect the high rate of IAV replication in the lungs of lymphocyte-deficient hosts, and therefore their disease-ameliorating effect was mediated through the suppression of innate immune pathology. Mechanistically, Treg cells reduced the accumulation and altered the distribution of monocytes/macrophages in the lungs of IAV-infected hosts. This reduction in lung monocytosis was associated with a specific delay in monocyte chemotactic protein-2 (MCP-2) induction in the infected lungs. Nevertheless, Treg cells failed to prevent the eventual development of severe disease in lymphocyte-deficient hosts, which likely was caused by the ongoing IAV replication. Indeed, using T-cell-deficient mice, which mounted a T-cell-independent B cell response to IAV, we further showed that the combination of virus-neutralizing antibodies and transferred Treg cells led to the complete prevention of clinical disease following IAV infection. Taken together, these results suggested that innate immune pathology and virus-induced pathology are the two main contributors to pathogenesis during IAV infection.

Title: Roosting ecology and variation in adaptive and innate immune system function in the Brazilian free-tailed bat (Tadarida brasiliensis) Content: Bats have recently been implicated as reservoirs of important emerging diseases. However, few studies have examined immune responses in bats, and even fewer have evaluated these responses in an ecological context. We examined aspects of both innate and adaptive immune response in adult female Brazilian free-tailed bats (Tadarida brasiliensis) at four maternity roosts (two natural caves and two human-made bridges) in south-central Texas. Immune measurements included in vitro bactericidal ability of whole blood and in vivo T cell mediated response to mitogenic challenge. Bactericidal activity in T. brasiliensis varied with roosting ecology, but appears to be sensitive to colony-level effects. Blood from females living at one cave had significantly lower bactericidal ability than blood from females at three other sites. T cell mediated response in this species was associated with variation in roost ecology, with females from two caves having greater responses than females from two bridges. T cell mediated response and bactericidal activity were negatively correlated with one another within individuals that were tested for both. Variation in immunological response of T. brasiliensis is important for understanding the influence of the environment on the frequency and distribution of immunologically competent individuals and for understanding disease-host dynamics in this and other colonial species.

Title: Immune response to SARS‐CoV‐2 and mechanisms of immunopathological changes in COVID‐19 Content: As a zoonotic disease that has already spread globally to several million human beings and possibly to domestic and wild animals, eradication of coronavirus disease 2019 (COVID‐19) appears practically impossible. There is a pressing need to improve our understanding of the immunology of this disease to contain the pandemic by developing vaccines and medicines for the prevention and treatment of patients. In this review, we aim to improve our understanding on the immune response and immunopathological changes in patients linked to detoriating clinical conditions such as, cytokine storm, acute respiratory distress syndrome, autopsy findings and changes in acute phase reactants and serum biochemistry in COVID‐19. Similar to many other viral infections, asymptomatic disease is present in a significant but currently unknown fraction of the affected individuals.In the majority of the patients, a one‐week, self‐limiting viral respiratory disease typically occurs, which ends with the development of neutralizing anti‐viral T cell and antibody immunity. The IgM, IgA and IgG type virus‐specific antibodies levels are important measurements to predict population immunity against this disease and whether cross‐reactivity with other coronaviruses is taking place.High viral‐load during the first infection and repeated exposure to virus especially in healthcare workers can be an important factor for severity of disease. It should be noted that many aspects of severe patients are unique to COVID‐19 and are rarely observed in other respiratory viral infections, such as severe lymphopenia and eosinopenia, extensive pneumonia and lung tissue damage, a cytokine storm leading to acute respiratory distress syndrome and multiorgan failure. Lymphopenia causes a defect in antiviral and immune regulatory immunity. At the same time, a cytokine storm starts with extensive activation of cytokine‐secreting cells with innate and adaptive immune mechanisms both of with contribute to a poor prognosis. Elevated levels of acute phase reactants and lymphopenia are early predictors of high disease severity. Prevention of development to severe disease, cytokine storm, acute respiratory distress syndrome and novel approachs to prevent their development will be main routes for future research areas. As we learn to live amidst the virus, understanding the immunology of the disease can assist in containing the pandemic and in developing vaccines and medicines to prevent and treat individual patients.

Title: Immune Responses to Middle East Respiratory Syndrome Coronavirus During the Acute and Convalescent Phases of Human Infection Content: BACKGROUND: An understanding of immune responses against the Middle East respiratory syndrome (MERS) is important for the development of treatments and preventive measures. Here, we investigated the spectrum of immune responses occurring in patients with MERS during the early period of infection. METHODS: We obtained peripheral blood samples from 27 hospitalized patients recruited during the epidemic that occurred in 2015 in South Korea. Plasma cytokines/chemokines and antibodies were quantified. Virus-specific T cells were examined by intracellular cytokine staining after stimulation of peripheral blood mononuclear cells with overlapping peptides spanning whole virus structural proteins. RESULTS: At the acute phase of infection, elevated levels of plasma proinflammatory cytokines/chemokines were detected in proportion to the severity of the disease. Distinctively high frequencies of MERS coronavirus–reactive CD8(+) T cells were also observed in patients with severe/moderate illness, whereas antibody and CD4(+) T-cell responses were minimally detected at this stage. At the convalescent phase, disease severity–dependent antibody responses emerged and antigen-reactive cells were identified in both T-cell subsets. These T cells belonged to the T-helper 1 or type 1 cytotoxic T cell subtypes. While CD8(+) T cells responded preferentially to the viral S protein compared with E/M/N proteins, especially at the acute stage, slightly more CD4(+) T cells recognized E/M/N proteins compared with S protein at the convalescent phase. CONCLUSIONS: Our findings show an association between the early CD8(+) T-cell response and the severity of the infection, and also provide basic information that may help to prepare effective control strategies for MERS in humans.

Title: Expansion of SARS-CoV-2-specific Antibody-secreting Cells and Generation of Neutralizing Antibodies in Hospitalized COVID-19 Patients Content: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 and has since become a global pandemic. Pathogen-specific antibodies are typically a major predictor of protective immunity, yet B cell and antibody responses during COVID-19 are not fully understood. Here, we analyzed antibody-secreting cell (ASC) and antibody responses in twenty hospitalized COVID-19 patients. The patients exhibited typical symptoms of COVID-19, and presented with reduced lymphocyte numbers and increased T cell and B cell activation. Importantly, we detected an expansion of SARS-CoV-2 nucleocapsid protein-specific ASCs in all twenty COVID-19 patients using a multicolor FluoroSpot assay. Out of the 20 patients, 16 had developed SARS-CoV-2-neutralizing antibodies by the time of inclusion in the study. SARS-CoV-2-specific IgA, IgG and IgM antibody levels positively correlated with SARS-CoV-2-neutralizing antibody titers, suggesting that SARS-CoV-2-specific antibody levels may reflect the titers of neutralizing antibodies in COVID-19 patients during the acute phase of infection. Lastly, we showed that interleukin 6 (IL-6) and C-reactive protein (CRP) concentrations were higher in serum of patients who were hospitalized for longer, supporting the recent observations that IL-6 and CRP could be used to predict COVID-19 severity. Altogether, this study constitutes a detailed description of clinical and immunological parameters in twenty COVID-19 patients, with a focus on B cell and antibody responses, and provides tools to study immune responses to SARS-CoV-2 infection and vaccination.

Title: Combination anti-HIV-1 antibody therapy is associated with increased virus-specific T cell immunity Content: Combination antiretroviral therapy (ART) is highly effective in controlling human immunodeficiency virus (HIV)-1 but requires lifelong medication due to the existence of a latent viral reservoir(1,2). Potent broadly neutralizing antibodies (bNAbs) represent a potential alternative or adjuvant to ART. In addition to suppressing viremia, bNAbs may have T cell immunomodulatory effects as seen for other forms of immunotherapy(3). However, this has not been established in individuals who are infected with HIV-1. Here, we document increased HIV-1 Gag-specific CD8(+) T cell responses in the peripheral blood of all nine study participants who were infected with HIV-1 with suppressed blood viremia, while receiving bNAb therapy during ART interruption(4). Increased CD4(+) T cell responses were detected in eight individuals. The increased T cell responses were due both to newly detectable reactivity to HIV-1 Gag epitopes and the expansion of pre-existing measurable responses. These data demonstrate that bNAb therapy during ART interruption is associated with enhanced HIV-1-specific T cell responses. Whether these augmented T cell responses can contribute to bNAb-mediated viral control remains to be determined.

Title: Pre-existing neutralizing antibodies prevent CD8 T cell-mediated immunopathology following respiratory syncytial virus infection Content: Despite being a leading cause of severe respiratory disease, there remains no licensed respiratory syncytial virus (RSV) vaccine. Neutralizing antibodies reduce the severity of RSV-associated disease, but are not sufficient for preventing reinfection. In contrast, the role of memory CD8 T cells in protecting against a secondary RSV infection is less established. We recently demonstrated that high-magnitude memory CD8 T cells efficiently reduced lung viral titers following RSV infection, but induced fatal immunopathology that was mediated by IFN-γ. To evaluate the ability of RSV-specific neutralizing antibodies to prevent memory CD8 T cell-mediated immunopathology, mice with high-magnitude memory CD8 T cell responses were treated with neutralizing antibodies prior to RSV challenge. Neutralizing antibody treatment significantly reduced morbidity and prevented mortality following RSV challenge compared with IgG-treated controls. Neutralizing antibody treatment restricted early virus replication, which caused a substantial reduction in memory CD8 T cell activation and IFN-γ production, directly resulting in survival. In contrast, therapeutic neutralizing antibody administration did not impact morbidity, mortality, or IFN-γ levels, despite significantly reducing lung viral titers. Therefore, only pre-existing neutralizing antibodies prevent memory CD8 T cell-mediated immunopathology following RSV infection. Overall, our results have important implications for the development of future RSV vaccines.

Title: Passive immunization does not provide protection against experimental infection with Mycoplasma haemofelis Content: Mycoplasma haemofelis (Mhf) is the most pathogenic feline hemotropic mycoplasma. Cats infected with Mhf that clear bacteremia are protected from Mhf reinfection, but the mechanisms of protective immunity are unresolved. In the present study we investigated whether the passive transfer of antibodies from Mhf-recovered cats to naïve recipient cats provided protection against bacteremia and clinical disease following homologous challenge with Mhf; moreover, we characterized the immune response in the recipient cats. Ten specified pathogen-free (SPF) cats were transfused with pooled plasma from cats that had cleared Mhf bacteremia; five control cats received plasma from naïve SPF cats. After homologous challenge with Mhf, cats were monitored for 100 days using quantitative PCR, hematology, blood biochemistry, Coombs testing, flow cytometry, DnaK ELISA, and red blood cell (RBC) osmotic fragility (OF) measurement. Passively immunized cats were not protected against Mhf infection but, compared to control cats, showed significantly higher RBC OF and B lymphocyte (CD45R/B220(+)) counts and occasionally higher lymphocyte, monocyte and activated CD4(+) T lymphocyte (CD4(+)CD25(+)) counts; they also showed higher bilirubin, total protein and globulin levels compared to those of control cats. At times of peak bacteremia, a decrease in eosinophils and lymphocytes, as well as subsets thereof (B lymphocytes and CD5(+), CD4(+) and CD8(+) T lymphocytes), and an increase in monocytes were particularly significant in the passively immunized cats. In conclusion, passive immunization does not prevent bacteremia and clinical disease following homologous challenge with Mhf, but enhances RBC osmotic fragility and induces a pronounced immune response. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13567-016-0361-x) contains supplementary material, which is available to authorized users.

Title: Vitamin E and immune response in the aged: molecular mechanisms and clinical implications Content: Summary: Nutritional status has been indicated as a contributing factor to age‐related dysregulation of the immune response. Vitamin E, a lipid‐soluble antioxidant vitamin, is important for normal function of the immune cells. The elderly are at a greater risk for vitamin E intake that is lower than recommended levels. Vitamin E supplementation above currently recommended levels has been shown to improve immune functions in the aged including delayed‐type hypersensitivity skin response and antibody production in response to vaccination, which was shown to be mediated through increased production of interleukin (IL)‐2, leading to enhanced proliferation of T cells, and through reduced production of prostaglandin E(2), a T‐cell suppressive factor, as a result of a decreased peroxynitrite formation. Vitamin E increased both cell‐dividing and IL‐producing capacities of naïve T cells, but not memory T cells. The vitamin E‐induced enhancement of immune functions in the aged was associated with significant improvement in resistance to influenza infection in aged mice and a reduced risk of acquiring upper respiratory infections in nursing home residents. Further studies are needed to determine the signaling mechanisms involved in the upregulation of naïve T‐cell function by vitamin E as well as the specific mechanisms involved in reduction of risk for upper respiratory infections.

Title: Virus-specific antibody, in the absence of T cells, mediates demyelination in mice infected with a neurotropic coronavirus. Content: Mice infected with mouse hepatitis virus strain JHM develop an inflammatory demyelinating disease in the central nervous system with many similarities to human multiple sclerosis. The mouse disease is primarily immune-mediated because demyelination is not detected in JHM-infected mice lacking T or B cells but does occur after transfer of JHM-specific T cells. Although less is known about the ability of antibodies to mediate demyelination, the presence of oligoclonally expanded B cells and high concentrations of antibodies (against self or infectious agents) in the central nervous system of many multiple sclerosis patients suggests that antibodies may also contribute to myelin destruction. Here, we show that anti-JHM antibodies, in the absence of T or B cells, caused demyelination in JHM-infected mice. Anti-JHM antibody was detected adjacent to areas of demyelination, consistent with a direct interaction between antibody and infected cells. Demyelination was reduced by 85 to 90% in infected RAG1(-/-) mice lacking normal expression of activating Fc receptors (FcRgamma(-/-)) and by approximately 76% when complement was depleted by treatment with cobra venom factor. These data demonstrate that JHM-specific antibodies are sufficient to cause demyelination and that myelin destruction in the presence of anti-virus antibodies results from a combination of complement- and Fc receptor-dependent mechanisms.

Title: Virus-Specific Antibody, in the Absence of T Cells, Mediates Demyelination in Mice Infected with a Neurotropic Coronavirus Content: Mice infected with mouse hepatitis virus strain JHM develop an inflammatory demyelinating disease in the central nervous system with many similarities to human multiple sclerosis. The mouse disease is primarily immune-mediated because demyelination is not detected in JHM-infected mice lacking T or B cells but does occur after transfer of JHM-specific T cells. Although less is known about the ability of antibodies to mediate demyelination, the presence of oligoclonally expanded B cells and high concentrations of antibodies (against self or infectious agents) in the central nervous system of many multiple sclerosis patients suggests that antibodies may also contribute to myelin destruction. Here, we show that anti-JHM antibodies, in the absence of T or B cells, caused demyelination in JHM-infected mice. Anti-JHM antibody was detected adjacent to areas of demyelination, consistent with a direct interaction between antibody and infected cells. Demyelination was reduced by 85 to 90% in infected RAG1−/− mice lacking normal expression of activating Fc receptors (FcRγ−/−) and by ∼76% when complement was depleted by treatment with cobra venom factor. These data demonstrate that JHM-specific antibodies are sufficient to cause demyelination and that myelin destruction in the presence of anti-virus antibodies results from a combination of complement- and Fc receptor-dependent mechanisms.

Title: Evaluation of Burkholderia mallei ΔtonB Δhcp1 (CLH001) as a live attenuated vaccine in murine models of glanders and melioidosis. Content: BACKGROUND Glanders caused by Burkholderia mallei is a re-emerging zoonotic disease affecting solipeds and humans. Furthermore, B. mallei is genetically related to B. pseudomallei, which is the causative agent of melioidosis. Both facultative intracellular bacteria are classified as tier 1 select biothreat agents. Our previous study with a B. mallei ΔtonB Δhcp1 (CLH001) live-attenuated vaccine demonstrated that it is attenuated, safe and protective against B. mallei wild-type strains in the susceptible BALB/c mouse model. METHODOLOGY/PRINCIPAL FINDING In our current work, we evaluated the protective efficacy of CLH001 against glanders and melioidosis in the more disease-resistant C57BL/6 mouse strain. The humoral as well as cellular immune responses were also examined. We found that CLH001-immunized mice showed 100% survival against intranasal and aerosol challenge with B. mallei ATCC 23344. Moreover, this vaccine also afforded significant cross-protection against B. pseudomallei K96243, with low level bacterial burden detected in organs. Immunization with a prime and boost regimen of CLH001 induced significantly greater levels of total and subclasses of IgG, and generated antigen-specific splenocyte production of IFN-γ and IL-17A. Interestingly, protection induced by CLH001 is primarily dependent on humoral immunity, while CD4+ and CD8+ T cells played a less critical protective role. CONCLUSIONS/SIGNIFICANCE Our data indicate that CLH001 serves as an effective live attenuated vaccine to prevent glanders and melioidosis. The quantity and quality of antibody responses as well as improving cell-mediated immune responses following vaccination need to be further investigated prior to advancement to preclinical studies.

Title: Immunologic characterization of a immunosuppressed multiple sclerosis patient that recovered from SARS-CoV-2 infection Content: A multiple sclerosis patient infected by SARS-CoV-2 during fingolimod therapy was hospitalized with moderate clinical features, and recovered in 15 days. High levels of CCL5 and CCL10 chemokines and of antibody-secreting B cells were detected, while the levels other B- and T-cell subsets were comparable to that of appropriate controls. However, CD4+ and CD8+ cells were oligoclonally expanded and prone to apoptosis when stimulated in vitro. This study suggests that fingolimod-immunosuppressed patients, despite the low circulating lymphocytes, may rapidly expand antibody-secreting cells and mount an effective immune response that favors COVID-19 recovery after discontinuation.

Title: Immunologic characterization of a immunosuppressed multiple sclerosis patient that recovered from SARS-CoV-2 infection Content: A multiple sclerosis patient infected by SARS-CoV-2 during fingolimod therapy was hospitalized with moderate clinical features, and recovered in 15 days. High levels of CCL5 and CCL10 chemokines and of antibody-secreting B cells were detected, while the levels other B- and T-cell subsets were comparable to that of appropriate controls. However, CD4+ and CD8+ cells were oligoclonally expanded and prone to apoptosis when stimulated in vitro. This study suggests that fingolimod-immunosuppressed patients, despite the low circulating lymphocytes, may rapidly expand antibody-secreting cells and mount an effective immune response that favors COVID-19 recovery after drug discontinuation.

Title: Minimal within-host dengue models highlight the specific roles of the immune response in primary and secondary dengue infections Content: In recent years, the within-host viral dynamics of dengue infections have been increasingly characterized, and the relationship between aspects of these dynamics and the manifestation of severe disease has been increasingly probed. Despite this progress, there are few mathematical models of within-host dengue dynamics, and the ones that exist focus primarily on the general role of immune cells in the clearance of infected cells, while neglecting other components of the immune response in limiting viraemia. Here, by considering a suite of mathematical within-host dengue models of increasing complexity, we aim to isolate the critical components of the innate and the adaptive immune response that suffice in the reproduction of several well-characterized features of primary and secondary dengue infections. By building up from a simple target cell limited model, we show that only the innate immune response is needed to recover the characteristic features of a primary symptomatic dengue infection, while a higher rate of viral infectivity (indicative of antibody-dependent enhancement) and infected cell clearance by T cells are further needed to recover the characteristic features of a secondary dengue infection. We show that these minimal models can reproduce the increased risk of disease associated with secondary heterologous infections that arises as a result of a cytokine storm, and, further, that they are consistent with virological indicators that predict the onset of severe disease, such as the magnitude of peak viraemia, time to peak viral load, and viral clearance rate. Finally, we show that the effectiveness of these virological indicators to predict the onset of severe disease depends on the contribution of T cells in fuelling the cytokine storm.

Title: Single-cell RNA-seq and V(D)J profiling of immune cells in COVID-19 patients Content: Coronavirus disease 2019 (COVID-19) has caused over 220,000 deaths so far and is still an ongoing global health problem. However, the immunopathological changes of key types of immune cells during and after virus infection remain unclear. Here, we enriched CD3+ and CD19+ lymphocytes from peripheral blood mononuclear cells of COVID-19 patients (severe patients and recovered patients at early or late stages) and healthy people (SARS-CoV-2 negative) and revealed transcriptional profiles and changes in these lymphocytes by comprehensive single-cell transcriptome and V(D)J recombination analyses. We found that although the T lymphocytes were decreased in the blood of patients with virus infection, the remaining T cells still highly expressed inflammatory genes and persisted for a while after recovery in patients. We also observed the potential transition from effector CD8 T cells to central memory T cells in recovered patients at the late stage. Among B lymphocytes, we analyzed the expansion trajectory of a subtype of plasma cells in severe COVID-19 patients and traced the source as atypical memory B cells (AMBCs). Additional BCR and TCR analyses revealed a high level of clonal expansion in patients with severe COVID-19, especially of B lymphocytes, and the clonally expanded B cells highly expressed genes related to inflammatory responses and lymphocyte activation. V-J gene usage and clonal types of higher frequency in COVID-19 patients were also summarized. Taken together, our results provide crucial insights into the immune response against patients with severe COVID-19 and recovered patients and valuable information for the development of vaccines and therapeutic strategies.

Title: Memory T cell responses targeting the SARS coronavirus persist up to 11 years post-infection Content: Severe acute respiratory syndrome (SARS) is a highly contagious infectious disease which first emerged in late 2002, caused by a then novel human coronavirus, SARS coronavirus (SARS-CoV). The virus is believed to have originated from bats and transmitted to human through intermediate animals such as civet cats. The re-emergence of SARS-CoV remains a valid concern due to the continual persistence of zoonotic SARS-CoVs and SARS-like CoVs (SL-CoVs) in bat reservoirs. In this study, the screening for the presence of SARS-specific T cells in a cohort of three SARS-recovered individuals at 9 and 11 years post-infection was carried out, and all memory T cell responses detected target the SARS-CoV structural proteins. Two CD8(+) T cell responses targeting the SARS-CoV membrane (M) and nucleocapsid (N) proteins were characterized by determining their HLA restriction and minimal T cell epitope regions. Furthermore, these responses were found to persist up to 11 years post-infection. An absence of cross-reactivity of these CD8(+) T cell responses against the newly-emerged Middle East respiratory syndrome coronavirus (MERS-CoV) was also demonstrated. The knowledge of the persistence of SARS-specific celullar immunity targeting the viral structural proteins in SARS-recovered individuals is important in the design and development of SARS vaccines, which are currently unavailable.

Title: Virus-Induced T Cell-Mediated Heterologous Immunity and Vaccine Development Content: Heterologous immunity (H.I.) is a consequence of an encounter with a specific antigen, which can alter the subsequent immune response to a different antigen. This can happen at the innate immune system level—often called trained immunity or innate immune memory—and/or at the adaptive immune system level involving T memory cells and antibodies. Viruses may also induce T cell-mediated H.I., which can confer protection or drive immunopathology against other virus subtypes, related or unrelated viruses, other pathogens, auto- or allo-antigens. It is important to understand the underlying mechanisms for the development of antiviral “universal” vaccines and broader T cell responses rather than just subtype-specific antibody responses as in the case of influenza. Furthermore, knowledge about determinants of vaccine-mediated H.I. may inform public health policies and provide suggestions for repurposing existing vaccines. Here, we introduce H.I. and provide an overview of evidence on virus- and antiviral vaccine-induced T cell-mediated cross-reactive responses. We also discuss the factors influencing final clinical outcome of virus-mediated H.I. as well as non-specific beneficial effects of live attenuated antiviral vaccines such as measles and vaccinia. Available epidemiological and mechanistic data have implications both for the development of new vaccines and for personalized vaccinology, which are presented. Finally, we formulate future research priorities and opportunities.

Title: Lactate dehydrogenase-elevating virus: an ideal persistent virus? Content: LDV contradicts all commonly held views about mechanisms of virus persistence, namely that persistence is primarily associated with noncytopathic viruses, or the selection of immune escape variants or other mutants, or a decrease in expression of certain viral proteins by infected cells, or replication in “immune-privileged sites”, or a general suppression of the host immune system, etc. [1, 2, 5, 54, 77, 78]. LDV is a highly cytocidal virus that invariably establishes a life-long, viremic, persistence in mice, in spite of normal anti-viral immune responses. One secret of LDV's success in persistence is its specificity for a renewable, nonessential population of cells that is continuously regenerated, namely a subpopulation of macrophages. Since the continuous destruction of these cells is not associated with any obvious health effects, this macrophage population seems nonessential to the well-being of its host. The only function identified for this subpopulation of macrophages is clearance of the muscle type of LDH and some other enzymes [59, 67, 68]. Furthermore, the effects of LDV infection on the host immune system, namely the polyclonal activation of B cells and its associated production of autoantibodies, and the slight impairment of primary and secondary antibody responses also do not seem to be severe enough to cause any clinical consequences. But how does LDV replication in macrophages escape all host defenses? Persistence is not dependent on the seletion of immune escape variants or other mutants ([58] and Palmer, Even and Plagemann, unpublished results). Also, LDV replication is not restricted to immune-privileged sites [5]. LDV replication persists in the liver, lymphoidal tissues and testis [66]. Only the latter could be considered a site not readily accessible to immune surveillance. Most likely, resistance of LDV replication to antiviral immune responses is related to the unique structure of its envelope proteins and the production of large quantities of viral antigens. High titers of anti-LDV antibodies are generated in infected mice but they neutralize LDV infectivity only very inefficiently and, even though the antiviral antibodies are mainly of the IgG2a and IgG2b isotypes, they do not mediate complement lyses of virions [31]. Interaction of the antibodies and complement with the VP-3/VP-2 heterodimers in the viral envelope may be impeded by the exposure of only very short peptide segments of these proteins at the envelope surface and the presence of large oligosaccharide side chains. Furthermore, since LDV maturation is restricted to intracytoplasmic cisternae [59, 71], the question arises of whether any of the viral proteins are available on the surface of infected cells for ADCC. CTLs also fail to control LDV replication. Altough CTLs specific for N/VP-1 are rapidly generated, these have disappeared by 30 days p.i. [26]. The reasons for this loss are unknown, but high-dose clonal exhaustion [41, 51, 77, 78] is a reasonable possibility since, regardless of the infectious dose, large amounts of LDV proteins are present in all the lymphoidal tissues at the time of the induction of the CTL response. Furthermore, after exhaustion of CTLs in the periphery, continuous replication of LDV in the thymus [65] assures that the mice become permanently immunologically tolerant with respect to LDV antigen-specific CTLs as a result of negative selection in the thymus. LDV might be a primary example for the effectiveness of a permanent clonal CTL deletion in adult animals under natural conditions of infection. The presumed modes of transmission of LDV in nature and the events associated with its infection of mice are strikingly similar to those observed during the acute and asymptomatic phases of infection with human immunodeficiency virus (HIV) [24, 29, 74, 78]. These include: (1) primary inefficient transmission via sexual and transplacental routes but effective transmission via blood; (2) primary replication in renewable populations of lymphoidal cells with production of large amounts of virus after the initial infection of the host followed by persistent low level of viremia in spite of antiviral immune responses; (3) persistence, reflecting continuous rounds of productive, cytocidal infection of permissive cells [59, 74] and the rate of generation of permissive cells which may be the main factor in determining the level of virus production (in the case of HIV, the rate of activation of CD4(+) T cells to support a productive HIV replication might be the factor determining the rate of virus production and the progression of the disease); (4) rapid antibody formation but delayed production of neutralizing antibodies with limited neutralizing capacity; (5) rapid but transient generation of virus-specific CTLs; and (6) accumulation of large amounts of virus in newly formed germinal centers in the spleen and lymph nodes concomitant with an initiation of a permanent polyclonal activation of B cells resulting in an elevation of plasma IgG2a. The events described under points 2–6 might be generally associated with natural viremic persistent virus infections. Such persistent viruses, by necessity, have evolved properties that allow them to escape all host defenses and control of their infection by immunological processes is, therefore, difficult, if not impossible. Prevention of infection and chemotherapy may be the only approaches available to combat such virus infections.

Title: Heterologous immunity between viruses. Content: Immune memory responses to previously encountered pathogens can sometimes alter the immune response to and the course of infection of an unrelated pathogen by a process known as heterologous immunity. This response can lead to enhanced or diminished protective immunity and altered immunopathology. Here, we discuss the nature of T-cell cross-reactivity and describe matrices of epitopes from different viruses eliciting cross-reactive CD8(+) T-cell responses. We examine the parameters of heterologous immunity mediated by these cross-reactive T cells during viral infections in mice and humans. We show that heterologous immunity can disrupt T-cell memory pools, alter the complexity of the T-cell repertoire, change patterns of T-cell immunodominance, lead to the selection of viral epitope-escape variants, alter the pathogenesis of viral infections, and, by virtue of the private specificity of T-cell repertoires within individuals, contribute to dramatic variations in viral disease. We propose that heterologous immunity is an important factor in resistance to and variations of human viral infections and that issues of heterologous immunity should be considered in the design of vaccines.

Title: Targeted Deletion of FGL2 Leads to Increased Early Viral Replication and Enhanced Adaptive Immunity in a Murine Model of Acute Viral Hepatitis Caused by LCMV WE Content: Mounting effective innate and adaptive immune responses are critical for viral clearance and the generation of long lasting immunity. It is known that production of inhibitory factors may result in the inability of the host to clear viruses, resulting in chronic viral persistence. Fibrinogen-like protein 2 (FGL2) has been identified as a novel effector molecule of CD4(+)CD25(+) Foxp3(+) regulatory T (Treg) cells that inhibits immune activity by binding to FCγRIIB expressed primarily on antigen presenting cells (APC). In this study, we show that infection of mice with Lymphocytic Choriomeningitis Virus WE (LCMV WE) leads to increased plasma levels of FGL2, which were detected as early as 2 days post-infection (pi) and persisted until day 50 pi. Mice deficient in FGL2 (fgl2(−/−)) had increased viral titers of LCMV WE in the liver early p.i but cleared the virus by day 12 similar to wild type mice. Dendritic cells (DC) isolated from the spleens of LCMV WE infected fgl2(−/−) had increased expression of the DC maturation markers CD80 and MHC Class II compared to wild type (fgl2(+/+)). Frequencies of CD8(+) and CD4(+) T cells producing IFNγ in response to ex vivo peptide re-stimulation isolated from the spleen and lymph nodes were also increased in LCMV WE infected fgl2 (−/−) mice. Increased frequencies of CD8(+) T cells specific for LCMV tetramers GP(33) and NP(396) were detected within the liver of fgl2(−/−) mice. Plasma from fgl2(−/−) mice contained higher titers of total and neutralizing anti-LCMV antibody. Enhanced anti-viral immunity in fgl2(−/−) mice was associated with increased levels of serum alanine transaminase (ALT), hepatic necrosis and inflammation following LCMV WE infection. These data demonstrate that targeting FGL2 leads to early increased viral replication but enhanced anti-viral adaptive T & B cell responses. Targeting FGL2 may enhance the efficacy of current anti-viral therapies for hepatotropic viruses.

Title: A Crucial Role for Infected-Cell/Antibody Immune Complexes in the Enhancement of Endogenous Antiviral Immunity by Short Passive Immunotherapy Content: Antiviral monoclonal antibodies (mAbs) represent promising therapeutics. However, most mAbs-based immunotherapies conducted so far have only considered the blunting of viral propagation and not other possible therapeutic effects independent of virus neutralization, namely the modulation of the endogenous immune response. As induction of long-term antiviral immunity still remains a paramount challenge for treating chronic infections, we have asked here whether neutralizing mAbs can, in addition to blunting viral propagation, exert immunomodulatory effects with protective outcomes. Supporting this idea, we report here that mice infected with the FrCas(E) murine retrovirus on day 8 after birth die of leukemia within 4–5 months and mount a non-protective immune response, whereas those rapidly subjected to short immunotherapy with a neutralizing mAb survive healthy and mount a long-lasting protective antiviral immunity with strong humoral and cellular immune responses. Interestingly, the administered mAb mediates lysis of infected cells through an antibody-dependent cell cytotoxicity (ADCC) mechanism. In addition, it forms immune complexes (ICs) with infected cells that enhance antiviral CTL responses through FcγR-mediated binding to dendritic cells (DCs). Importantly, the endogenous antiviral antibodies generated in mAb-treated mice also display the same properties, allowing containment of viral propagation and enhancement of memory cellular responses after disappearance of the administered mAb. Thus, our data demonstrate that neutralizing antiviral mAbs can act as immunomodulatory agents capable of stimulating a protective immunity lasting long after the end of the treatment. They also show an important role of infected-cells/antibody complexes in the induction and the maintenance of protective immunity through enhancement of both primary and memory antiviral T-cell responses. They also indicate that targeting infected cells, and not just viruses, by antibodies can be crucial for elicitation of efficient, long-lasting antiviral T-cell responses. This must be considered when designing antiviral mAb-based immunotherapies.

Title: Novel genetically-modified chimpanzee adenovirus and MVA-vectored respiratory syncytial virus vaccine safely boosts humoral and cellular immunity in healthy older adults Content: Abstract Objectives Respiratory syncytial virus (RSV) causes respiratory infection across the world, with infants and the elderly at particular risk of developing severe disease and death. The replication-defective chimpanzee adenovirus (PanAd3-RSV) and modified vaccinia virus Ankara (MVA-RSV) vaccines were shown to be safe and immunogenic in young healthy adults. Here we report an extension to this first-in-man vaccine trial to include healthy older adults aged 60–75 years. Methods We evaluated the safety and immunogenicity of a single dose of MVA-RSV given by intra-muscular (IM) injection (n = 6), two doses of IM PanAd3-RSV given 4-weeks apart (n = 6), IM PanAd3-RSV prime and IM MVA-RSV boost 8-weeks later (n = 6), intra-nasal (IN) spray of PanAd3-RSV prime and IM MVA-RSV boost 8-weeks later (n = 6), or no vaccine (n = 6). Safety measures included all adverse events within one week of vaccination and blood monitoring. Immunogenicity measures included serum antibody responses (RSV- and PanAd3-neutralising antibody titres measured by plaque-reduction neutralisation and SEAP assays, respectively), peripheral B-cell immune responses (frequencies of F-specific IgG and IgA antibody secreting cells and memory B-cells by ex vivo and cultured dual-colour ELISpot assays respectively), and peripheral RSV-specific T-cell immune responses (frequencies of IFNγ-producing T-cells by ex vivo ELISpot and CD4+/CD8+/Tfh-like cell frequencies by ICS/FACS assay). Results The vaccines were safe and well tolerated. Compared with each individual baseline immunity the mean fold-changes in serum RSV-neutralising antibody, appearance and magnitude of F-specific IgG and IgA ASCs and expansion of CD4+/CD8+ IFNγ-producing T-cells in peripheral circulation were comparable to the results seen from younger healthy adults who received the same vaccine combination and dose. There were little/no IgA memory B-cell responses in younger and older adults. Expansion of IFNγ-producing T-cells was most marked in older adults following IM prime, with balanced CD4+ and CD8+ T cell responses. The RSV-specific immune responses to vaccination did not appear to be attenuated in the presence of PanAd3 (vector) neutralising antibody. Conclusions PanAd3-RSV and MVA-RSV was safe and immunogenic in older adults and the parallel induction of RSV-specific humoral and cellular immunity merits further assessment in providing protection from severe disease.

Title: Protective Effects of Glutamine Antagonist 6-Diazo-5-Oxo-l-Norleucine in Mice with Alphavirus Encephalomyelitis. Content: UNLABELLED Inflammation is a necessary part of the response to infection but can also cause neuronal injury in both infectious and autoimmune diseases of the central nervous system (CNS). A neurovirulent strain of Sindbis virus (NSV) causes fatal paralysis in adult C57BL/6 mice during clearance of infectious virus from the CNS, and the virus-specific immune response is implicated as a mediator of neuronal damage. Previous studies have shown that survival is improved in T-cell-deficient mice and in mice with pharmacological inhibition of the inflammatory response and glutamate excitotoxicity. Because glutamine metabolism is important in the CNS for the generation of glutamate and in the immune system for lymphocyte proliferation, we tested the effect of the glutamine antagonist DON (6-diazo-5-oxo-l-norleucine) on the outcome of NSV infection in mice. DON treatment for 7 days from the time of infection delayed the onset of paralysis and death. Protection was associated with reduced lymphocyte proliferation in the draining cervical lymph nodes, decreased leukocyte infiltration into the CNS, lower levels of inflammatory cytokines, and delayed viral clearance. In vitro studies showed that DON inhibited stimulus-induced proliferation of lymphocytes. When in vivo treatment with DON was stopped, paralytic disease developed along with the inflammatory response and viral clearance. These studies show that fatal NSV-induced encephalomyelitis is immune mediated and that antagonists of glutamine metabolism can modulate the immune response and protect against virus-induced neuroinflammatory disease. IMPORTANCE Encephalomyelitis due to infection with mosquito-borne alphaviruses is an important cause of death and of long-term neurological disability in those who survive infection. This study demonstrates the role of the virus-induced immune response in the generation of neurological disease. DON, a glutamine antagonist, inhibited the proliferation of lymphocytes in response to infection, prevented the development of brain inflammation, and protected mice from paralysis and death during treatment. However, because DON inhibited the immune response to infection, clearance of the virus from the brain was also prevented. When treatment was stopped, the immune response was generated, brain inflammation occurred, virus was cleared, and mice developed paralysis and died. Therefore, more definitive treatment for alphaviral encephalomyelitis should inhibit virus replication as well as neuroinflammatory damage.

Title: Protective immunity after COVID-19 has been questioned: What can we do without SARS-CoV-2-IgG detection? Content: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces a severe acute respiratory syndrome that is called COVID-19. Clinical manifestations of COVID-19 include diarrhea, pneumonia, lymphopenia, exhausted lymphocytes, and pro-inflammatory cytokine production. Immunology is part of the process of clinical evolution, but there are some questions around immunity-based protection: (1) why some infected people have only mild symptoms of the disease or are asymptomatic; (2) why delayed and weak antibody responses are associated with severe outcomes; and (3) why positivity in molecular tests does not represent protective antibody IgG. Perhaps T cell responses may be the key to solving those questions. SARS-CoV-2-specific memory T cells persist in peripheral blood and may be capable of providing effective information about protective immunity. The T cells studies can be helpful in elucidating the pathways for development of vaccines, therapies, and diagnostics for COVID-19 and for filling these immunology knowledge gaps.

Title: Protective immunity after COVID-19 has been questioned: what can we do without SARS-CoV-2-IgG detection? Content: Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces a severe acute respiratory syndrome that is called COVID-19. Clinical manifestations of COVID-19 include diarrhea, pneumonia, lymphopenia, exhausted lymphocytes, and pro-inflammatory cytokine production. Immunology is part of the process of clinical evolution, but there are some questions around immunity-based protection: (1) why some infected people have only mild symptoms of the disease or are asymptomatic; (2) why delayed and weak antibody responses are associated with severe outcomes; and (3) why positivity in molecular tests does not represent protective antibody IgG. Perhaps T cell responses may be the key to solving those questions. SARS-CoV-2-specific memory T cells persist in peripheral blood and may be capable of providing effective information about protective immunity. The T cells studies can be helpful in elucidating the pathways for development of vaccines, therapies, and diagnostics for COVID-19 and for filling these immunology knowledge gaps.

Title: Immunologic perturbations in severe COVID-19/SARS-CoV-2 infection Content: Although critical illness has been associated with SARS-CoV-2-induced hyperinflammation, the immune correlates of severe COVID-19 remain unclear. Here, we comprehensively analyzed peripheral blood immune perturbations in 42 SARS-CoV-2 infected and recovered individuals. We identified broad changes in neutrophils, NK cells, and monocytes during severe COVID-19, suggesting excessive mobilization of innate lineages. We found marked activation within T and B cells, highly oligoclonal B cell populations, profound plasmablast expansion, and SARS-CoV-2-specific antibodies in many, but not all, severe COVID-19 cases. Despite this heterogeneity, we found selective clustering of severe COVID-19 cases through unbiased analysis of the aggregated immunological phenotypes. Our findings demonstrate broad immune perturbations spanning both innate and adaptive leukocytes that distinguish dysregulated host responses in severe SARS-CoV-2 infection and warrant therapeutic investigation. One Sentence Summary Broad immune perturbations in severe COVID-19