HIV SUPPRESSOR FACTOR DERIVED FROM SCRUB TYPHUS

Abstract: A suppressor factor obtained from the blood of a mammal infected with scrub typhus which has anti-HIV activity, antigens, antibodies and vaccines derived therefrom, and uses thereof for prevention and treatment of HIV infection.

Description: HIV SUPPRESSOR FACTOR DERIVED FROM SCRUB TYPHUS BACKGROUND OF THE INVENTION 1. Field of the Invention The invention relates to a suppressor factor for HIV derived from mammals infected with Orientia tsutsugamushi (scrub typhus), and to compositions containing the suppressor factor and derivatives thereof for prevention and treatment of HIV infection. 2. Background Information Human Acquired Immune Deficiency Syndrome (ALDS) and infection by human immunodeficiency virus (HIV), its causative agent, have become serious worldwide problems since their emergence in the last several decades. In 1998, the United Nations estimated that 5.8 million individuals become newly infected with HIV each year (1). The problem is particularly severe in developing countries. At present, there are no completely effective treatments for HIV infection or AIDS, although progress has been made in the development of antiretroviral drugs and drug combinations, which in some individuals have been effective in slowing the progress of the disease (2). Antiretroviral drugs are expensive and are often poorly tolerated. Furthermore, greater than 90% of those currently infected with the HIV-1 virus will never have access to effective antiretroviral drugs (1). Three or four antiretroviral drugs are often recommended both for treatment and also for prevention (post-exposure chemoprophylaxis) of HIV after a suspected high risk exposure (3). Therefore, a need exists for a safer and more economical method of treatment and chemoprophylaxis. O. tsutsugamushi is a small gram-negative bacillus that is distinct from other bacteria because of its obligate intracellular parasitism. It typically ranges in size from 1.0 to 2.0 mm by 0.3 mm, but pleomorphism is common. O. tsutsugamushi infection causes the disease called scrub typhus. The natural cycle of infection involves mites and small rodents. Humans are accidental hosts and acquire scrub typhus from the bite of an infected chigger. Neither direct mouse to mouse nor mouse to human transmission has been reported. Multiple serotypes of O. tsutsugamushi cause illness and produce effective homologous immunity but transient cross-protection. Signs and symptoms of scrub typhus vary from those of a mild, self-limiting illness to a fatal syndrome with failure of one or several major organ systems. An eschar at the site of the chigger bite is the most characteristic sign, but is not always present or goes unnoticed by the patient and physician. Direct injection of patient blood into mice remains the most sensitive method of isolating the causative organism, despite careful attempts at finding alternative methods. The present inventors have discovered that AIDS patients who become infected by some, but not all, strains of scrub typhus experience a marked reduction in HIV viral load, with the maximum inhibitory effect at 0-14 days after admission to hospital. Sera obtained from some HIV negative patients with acute scrub typhus reduces HIV viral infectivity in vitro. Some scrub typhus sera reduce infectivity even more than HIV antibody positive sera in vitro. The interaction between scrub typhus and HIV appears to be bidirectional. Scrub typhus is no more severe in HIV seropositive individuals, but it is more difficult to isolate O. tsutsugamushi from their blood (14% vs. 49% in HIV seronegative patients, Ref. 4). SUMMARY OF THE INVENTION This invention takes advantage of the anti-HIV effects of an inhibitory factor produced during scrub typhus infection. The present inventors have discovered that scrub typhus infection reduces HIV viral load in patients who are also infected with HIV-1. In addition, sera from scrub typhus patients who are HIV seronegative exhibit anti-HIV- 1 activity in vitro. Various scrub typhus strains were cultivated and tested to study this phenomenon further. Sera from mice infected with strains which exhibited such activity are strongly reactive with HIV-1 pl7 antigen peptides. It is an object of the invention to provide a method of inducing an immunogenic response to human immunodeficiency virus (HIV), in particular to HIV-1. This immunogenic response is induced by exploiting the fact that certain epitopes present in scrub typhus are identical or similar to those present in HIV. The invention includes inhibitory factors, antibodies, antigens, peptides, immunogenic compositions and vaccines useful for prophylaxis and/or treatment of HIV infection, as well as methods for their use. After a scrub typhus strain with anti-HIV activity has been identified, isolation and testing of peptides from the organisms, purification of plasma and serum, and isolation of antibody from blood to be used in the compositions and methods of the invention can be carried out by routine methods known to those of skill in the art. Accordingly, it is an object of the invention to provide a novel suppressor factor derived from scrub typhus infection (Orientia tsutsugamushi) which is useful for the prevention and treatment of HIV infection and AIDS. In particular, it is an object of the invention to provide a suppressor factor derived from scrub typhus infection in the form of purified sera, plasma or immunoglobulin which is suitable for administration to patients at risk for, or infected with, HIV, either alone or in combination with other agents. It is a particular object of the invention to provide an isolated suppressor factor from sera or plasma taken from patients with scrub typhus which has anti-HIV activity. By "anti-HIV" activity is meant an effect of inhibiting HIV replication in vitro, or of reducing HIV viral load in vivo. Such inhibition or reduction is considered to occur if replication or viral load is reduced by at least about 3-fold, and more preferably by more than 90%. Such serum may be that obtained from an individual patient, or may be pooled from two or more patients. As used herein, the term "isolated" is intended to mean separated from its natural environment. The term "purified" is intended to mean having naturally associated substances further removed, but not necessarily to 100% purity. Purified sera, plasma or immunoglobulin which contains the anti-HIV suppressor factor according to the invention can be obtained by identifying patients having the suppressor factor according to the methods set forth hereinbelow and isolating and purifying the sera, plasma or immunoglobulin according to suitable methods known in the art. It is also an object of the invention to provide peptides from inhibitory strains of scrub typhus. Such peptides function as antigens and can be used to produce antibodies effective for the prophylaxis and treatment of HIV infection. After isolation of inhibitory strains of scrub typhus, peptides useful for the invention can be made by routine methods which are familiar to those of skill in the art. The term "antigen", as used herein, includes such peptides, and particularly includes, mter alia, peptides from 6 to 100 and more preferably 12 to 72 amino acid residues in length which are capable of eliciting production of such antibodies. Such peptides can be made synthetically, for example, and screened for activity. The term "antibody", as used herein, includes, but is not limited to, polyclonal antibodies as well as monoclonal antibodies, which may be naturally derived from any animal, synthesized in bacteria, or chemically or genetically synthesized. The antibody may be a whole antibody or an antigen binding fragment thereof and may in general belong to any immunoglobulin class. Thus, for example, it may be an immunoglobulin M antibody or an immunoglobulin G antibody. It is a particular object of the invention to provide an IgM and/or IgG anti-scrub typhus antibody that recognizes HIV pi 7 antigen or p24 antigen. By "anti-scrub typhus antibody" is meant antibody which recognizes, binds to, and/or inhibits the action of scrub typhus organisms. Serum, plasma or antibodies can be tested to determine whether it contains antibodies recognizing HIV pi 7 antigen or p24 antigen by means known to those of skill in the art. Such recognition can be demonstrated by immunoblotting techniques and/or enzyme-linked immunoassay (ELISA), as set forth hereinbelow, or by other standard techniques known to those of skill in the art. It is a further object of the invention to provide scrub typhus antibodies which cross-react with HIV-1 proteins. By "cross-react with HIV proteins" is meant that the antibodies recognize and/or bind to HIV proteins. It is a further object of the invention to provide an antibody that inhibits HIV virus replication by binding directly to HIV or to HIV infected cells and affecting any or all of the steps involved in the viral replication cycle. It is a further object of the invention to provide a factor that suppresses HIV-1 virus replication. Such factor may be, for example, an antibody to scrub typhus which operates directly by binding to HIV, or otherwise by interfering in the replication process, or an antigen which stimulates production of such an antibody or antibodies. The peptides of the invention may be used to produce monoclonal antibodies using, for example, hybridoma technology. These monoclonal antibodies can be administered to HIV infected patients as treatment and/or injected at intervals to HIV seronegative individuals as a means for preventing HIV infection (passive immunization). The peptides could also be administered to HIV negative individuals in order to stimulate production of anti-HIV antibodies (active immunization). Thus, the invention is contemplated to include compositions containing such peptides, including, but not limited to, vaccines, and methods for their use in prevention and treatment of HIV infection. The compositions of the invention are also contemplated to be used to prevent maternal -fetal perinatal transmission of HIV. As used herein, the term "vaccine" intended to mean both prophylactic and therapeutic immunogenic compositions for prevention and treatment of HIV infection. Such immunogenic compositions may be active, containing an effective amount of a peptide according to the invention which is capable of eliciting in a host a protective immune response to HIV infection, along with a pharmaceutically acceptable diluent or carrier. Alternatively, such immunogenic compositions may be used to generate antibodies according to the invention which can be administered in an amount which is sufficient to confer a protective or therapeutic effect. The immunogenic compositions of the invention are administered in amounts readily determined by persons of ordinary skill in the art by routine experimentation. In vaccines, and when used otherwise for prophylaxis or therapy, the immunogenic compositions of the present invention may be used in conjunction with pharmaceutically acceptable adjuvants, carriers, diluents and excipients. In particular, the immunogenic composition may further comprise at least one adjuvant, such as aluminum phosphate or aluminum hydroxide. Suitable advujants, excipients, diluents or carriers will be known by those skilled in the art, and can be found in, for example, Remington's Pharmaceutical Sciences (18 th ed., 1990). The preferred means of administration compositions of the invention which are contemplated to be used for immunization and/or treatment is via intramuscular, intravenous or subdermal injection. However, they may be administered by any conventional means that are determined by those of skill in the art to be safe and effective, including oral and mucosal delivery. The invention differs from previous approaches to HIV therapy and prevention because it does not rely on working with HIV virus. Instead, it relies on the novel, cross-reactive inhibitory effects bestowed by an entirely different organism. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 (upper panel). HIV RNA copy numbers (expressed as percent of the day 28 measurement) are shown for the 10 individuals with scrub typhus (red circles) and the 5 patients with acute non-typhus infections (blue circles). The median viral loads (solid squares) of scrub typhus patients are connected by a red line and those of the control group by a blue line. Figure 1 (lower panel). Sequential HIV RNA concentration in plasma of the 4 patients coinfected with HIV and scrub typhus who had at least a 3-fold reduction in copy number relative to the day 28 value. RNA copy numbers were below the limits of detection in the 2 patients displayed in red; ST2 (day 14) and ST3 (days 0,3 and 7). Viral load was assayed by Roche Amplicor RT-PCR in one patient (blue dotted line) and by a bDNA assay in the others. Figure 2. LFA performed with HIV- 1 infected lymphocytes and sera taken 1 month after mice were infected with scrub typhus. The polar staining (top left X40) and bottom (XI 00) is not seen with preimmune mouse sera (top right X40). Figure 3. ELISA data at day 0 (presentation with scrub typhus), day 14 and day 600 post presentation for a scrub typhus patient who is not infected with HIV. P13, P41 and PI 05 are peptides derived from HIV P17 gag protein. The blue shows IgM reactivity and the red shows IgG reactivity. The data demonstrate that an HIV uninfected individual gains immune reactivity to epitopes on HIV proteins during scrub typhus infection. Figure 4. Scrub typhus infection causes mice to acquire stong antibodies to a gag pl7 epitope. The response at day 28 is IgM antibody. SV40 is one strain of scrub typhus. Figure 5. Reactivity to peptide 41 in groups of mice infected with 3 different strains of scrub typhus (240, 155, krp) and a control group. The serum reactivity of each group is compared to a control peptide from a different region of pl7 (pep 129) DETAILED DESCRIPTION OF THE INVENTION Informed consent is obtained from human subjects who receive full counseling for HIV testing under a protocol approved by the participating institutions located at Chiangrai Regional Hospital and Maesod Hospital (Ministry of Public Health, Thailand) and at the Armed Forces Research Institute of Medical Sciences, Bangkok (AFRIMS). Animal research is conducted in compliance with the Animal Welfare Act and animal use protocols were approved by the AFRIMS Institutional Animal Care and Use Committee. Example 1 Scrub typhus infection reduces HIV-1 viral load HIV-1 viral load was measured in patients infected with HIV-1 who were also diagnosed as having scrub typhus. The study was carried out as follows. Scrub typhus was initially diagnosed in adults with febrile illnesses of 1 week's duration or less and appropriate clinical features by dot-blot immunoassay (5) and confirmed by an indirect immunoperoxidase assay. Leptospirosis was diagnosed by an IgM specific ELISA (Panbio, Brisbane, Australia) (6) of proven specificity in Thailand (G. Watt; unpublished data). HIV-1 antibody was detected using two rapid screening tests (Serodia R -HIV, Fujirebio Inc., Tokyo, Japan, and Capillus R HIV- 1/ΗΓV-2, Cambridge Diagnostics, Galway, Ireland). Confirmatory HIV-1 Western blot (Novapath R HIV-1 Immunoblot, Bio-Rad, Hercules, California, USA) was performed if a screening test result was unclear or if there was a discrepancy between the results of 2 screening tests, in accordance with Thai government guidelines. HIV-1 RNA levels were measured in EDTA plasma using commercial kits (RT-PCR, Amplicor HIV Monitor test; Roche Molecular Systems, Branchburg, NJ,- limit of detection 50 copies/ml, CV 24%; and Chiron 2 nd generation bDNA assay, Chiron Corporation, Emeryville, CA- limit of detection 500 copies/ml, CV 15%). The same method was used for longitudinal assessment. CD4 counts were monitored using 2- color flow cytometry and a FACScan instrument (Becton Dickinson Immunocytometry Systems, San Jose, CA). HIV-1 subtyping and isolation HIV subtype was determined using peripheral blood mononuclear cells (PBMCs) from infected subjects and nested PCR employing primers which differentiate HIV subtypes B and E. (7) PBMCs from infected subjects were cocultured with phytohemagglutinin- stimulated PBMCs from a seronegative Thai donor at a 1 : 1 ratio. (7) The biological phenotype was determined on admission HIV isolates obtained from patients with <200 CD4+ cells. Supernatants from positive virus cultures were inoculated onto the CD4+ T cell line MT-2 using the technique described by the Division of AIDS, NIALD (8). Cultures were scored as either syncytium inducing (SI) or non-syncytium inducing (NSI). Follow-up of patients HIV seropositive subjects were evaluated clinically and by HIV viral loads 3, 7, 14 and 28 days after admission to the study. Patients were prospectively excluded from viral load analysis if there was clinical or laboratory suspicion of opportunistic or other infections, without knowledge of HIV viral load. PCR for HIV DNA was carried out on samples where viral load was below the limit of detection using primers to clade B and E viruses. Patients were treated with either chloramphenicol or doxycycline for scrub typhus, quinine or chloroquine for malaria, and penicillin for leptospirosis. The fever clearance time (FCT) was the number of hours between the first dose of medication and the first time when the morning oral temperature was < 37.2°C in the absence of antipyretic medication. Patients infected by HIV-1 were treated with the highest practically attainable standard of care in Thailand, which during the duration of this study did not include specific antiretroviral agents. An estimated 38,200 fever patients were screened during 12 study months at Chiangrai; fewer at Maesod. Scrub typhus was diagnosed in 173 patients; 22 of whom were known to be HIV seropositive. HIV serotesting identified an additional 19 subjects for inclusion. Thirty-one of these 41 patients were excluded from the final viral load analysis because additional pathogens were identified in blood or cerebrospinal fluid after enrollment (N=5), because clinical or laboratory findings were atypical of scrub typhus, malaria or leptospirosis (N=17), and because fever cleared initially but then recurred prior to day 28 (N=9). Longitudinal viral load measurements from 15 patients were included in the final analysis (Figure 1). Nine patients infected with O. tsutsugamushi, 1 with leptospirosis, and 1 with P. falciparum were studied in Chiangrai. Two patients with falciparum malaria, and one each with vivax malaria and scrub typhus were followed in Maesod. Only 4 of these 15 patients (27%) were previously known to be HIV seropositive. Admission characteristics of the 10 patients infected with O. tsutsugamushi were generally similar to admission characteristics of the 5 non-typhus infected individuals (Table 1). Table 1. Admission characteristics of the 15 HIV-1 infected subjects whose viral load measurements were included in the final analysis. The standard deviation is shown in parentheses for mean values, the range for median values. Scrub typhus Non-typhus N=10 malaria(4) leptospirosis(l) Demographic Information Age 31(±H) 41(±14) Male/female 6/4 3/2 Thai/hilltribe 6/4 5/0 Chiangrai/Maesod 9/1 2/3 Clinical Days of fever 4 (1-7) 3 (3-7) Oral temperature 38.2°C(±0.7) 38.2°C(±0.7) Hematocrit 34%(±6) 34%(±5) White blood cells/mm 5,915(1 ,420-27,390) 7,260(5,720-7,900) Platelets (XI , 000)/mm 3 120(63-357) 63(40-94) * CRP (mg/L) 40.9(0-146.2) 96.5(38.8-169.6) CD4 cells/mm 3 117(8-403) 255(153-299) HIV RNA (copies/ml) 59,220(0 -2,484,677) 384,740(10,000- 958,513) p<0.05 0 = below limit of detection of assay Platelet counts were significantly higher (p<0.05) in scrub typhus patients. CD4 cell counts and HIV-1 RNA copy numbers were lower, but not significantly so (p>0.1), in O. tsutsugamushi-m ' fected individuals. The median FCT in O. tsutsugamushi infection was 64h (range 41h-72h) and 66h (range 17h-67h) in the non-typhus group. The median viral load 3 days after admission was significantly lower in the scrub typhus group (193% vs 376%, p<0.05) as was the pooled median value for all loads measured from days 0-14 (108% vs 298%, p<0.05), compared with the non-typhus group. HIV RNA copy number fell below the threshold of detection in 2 individuals infected with O. tsutsugamushi, and 2 PBMC samples were also HIV DNA negative by PCR (Figure 1). Relative to the day 28 measurement, there was a 3-fold or greater reduction of viral load in 4 of the 10 patients with acute scrub typhus (2 received doxycycline and 2 received chloramphenicol) but in none of the non-typhus infected individuals (p=0.15). CD4+ lymphocytes were quantified 1 month after admission in 11 patients - 3 with a fall in viral load, 8 without. One month specimens were not collected from the other 4 subjects. CD4 cell counts were higher at 1 month than on admission by 166%, 174% and 52% in the 3 patients with a fall in copy number but fell by a median of 16% (range -42% to +56%) in the other 8 individuals (p <0.05). Primary HIV-1 viral isolates were obtained on admission from 17 patients with CD4 counts of < 200 cells/mm 3 , 6 of whom were included in the final viral load analysis (Table 2, numbers 1-6). Table 2. Phenotype of admission HIV-1 viral isolates (labelled 1-17), classified as either syncytia inducing (SI) or non-syncytia inducing (NSI). Phenotype CD4 cells/ml Scrub typhus 1. NSI 15 2. NSI 118 3. NSI 9 4. * NSI 161 5. NSI 116 6. NSI 66 7. NSI 107 NSI 7 9. NSI 131 10. NSI 147 Non-typhus 11. SI 17 12. SI 31 13. NSI 9 14. SI 25 15. SI 146 16. SI 22 17. NSI 175 subtype B, all others were subtype E. Five of the 7 isolates from non-typhus infected individuals were SI variants, but all 10 admission isolates obtained from O. tsutsugamushi-m ' fected patients were of the NSI phenotype (p<0.05). A single isolate (#4, Table 2) was HIV-1 subtype B, the remainder were subtype E virus. Acute phase reactants and immune activation. TNF-alpha was undetectable in admission sera from all 8 O. tsutsugamushi- infected patients tested (no specimens were available from 2 patients) and from 2 of 5 non-typhus infected individuals; concentrations were 24.0, 58.4 and 61.5 pg/ml in the other 3. TNF concentrations on day 28 were below detectable limits in all 8 scrub typhus and all 5 non-typhus specimens tested. There were no significant differences between median admission CRP concentrations in patients with scrub typhus (40.9 mg/L, range 0-146.2) and those without (96.5 mg/L, range 38.8-169.6; p>0.1). Day 28 CRP concentrations were below the limit of detection (5mg/L) in the 8 sera from the scrub typhus group and in 3 of 5 sera from non-typhus infected patients. Day 28 CRP concentrations in the other 2 malaria-infected subjects were 7.7 and 6.3 mg/L; CRP concentrations exceeding >8.0 mg L have been considered elevated in previous malaria studies (9). Macrophage inflamatory protein- 1 alpha (MLP-1 alpha) concentrations were below detectable limits and RANTES measurements were lower than the median reference value (49,137 pg/ml) and were within or below normal range in all 12 specimens assayed. RANTES concentrations did not correlate with viral load (r= - 0.26; p= 43). Plasma HIV-1 viral load fell by 3-fold or more in 4 of 10 patients not receiving antiretroviral therapy during acute scrub typhus infection (Figure 1). Four measurements were below the limits of detection and in 2 instances HIV-l DNA could not be detected by nested PCR (Figure 1, lower panel). No infectious agent has previously been demonstrated to reduce HIV viremia in ALDS patients. Rather, median plasma RNA copy numbers increased 7.8 fold during a prospective study of acute illness caused by a variety of pathogens (10) and transient elevations of viral load have been reported with intercurrent infection, immunization and even tuberculin skin testing (1 1 -14). The RNA copy number 3 days after enrollment was significantly lower in O. tsutsugamushi-m ' fected individuals than in the non-typhus group (p<0.05) even though a marked drop in viral load occurred in only a subset (40%) of scrub typhus patients. This suggested that inter-patient variability in HIV-1 suppression results from serologically unique epitopes of individual strains of O. tsutsugamushi (15). There was a significant association between scrub typhus and non-syncytia- inducing HIV variants (Table 2, p <0.001). NSI viruses are associated with delayed disease progression and slower viral replication (16). Scrub typhus may restrict HIV- induced syncytia formation by influencing pl7 protein, which is found in the membrane of HIV-infected lymphocytes and facilitates cell to cell spread of HIV-1 virus (17). Sera obtained during acute O. tsutsugamushi infection had potent HIV- neutralizing activity and was reactive with pl7 recombinant protein (Table 3). Regardless of mechanism, it is clear that and agent produced as a consequence of scrub typhus infection is effective in inducing immunogenicity to HIV. Table 3. Human (KDX5) and mouse (SV240) immunoassays. "ND" indicates not done. KDX5 a) Reduction of infectivity (% p24 antigen compared to normal human sera) PBMCs Day O Day 28 HIV pos 96h incubation 70% 48% -27% 120h incubation 73% 73% 2% H9 cells whole sera 92% 48% 100% antibody-depleted sera 42% 3% 66% b) pl7 ELISA (O.D.) serum dilution Day 14 Day 450 HIV pos 1:200 0.53 0.07 0.27 1:400 0.32 0.05 0.14 c) IgM peptide scanning (Sigma units) amino acids Day O Day 14 Day 28 Day 450 HIV pos 13-24 189.9 82.3 0 0 0 105-116 163.1 172.1 0 1 252.6 109-120 200.2 179.3 1.3 0 1.1 SV240 a) Syncytia per well (median - • 95%C.I.) Pre-immune Day 28 Experiment 1 101 (98-105) 48 (42-56) Experiment 2 145 (140-161) 76 (61-78) b) IgM pi 7 Western blot reactivity NO YES Peptide scanning of sera obtained early in infection revealed IgM reactivity at several regions within the pl7 protein. Convalescent sera (day 450) neither reacted with recombinant pl7 protein nor with synthetic pl7 peptides. Immune serum from scrub typhus-infected mice was reactive with the pl7 band on HIV Western blot, inhibited syncytia formation of HIV-infected lymphocytes, and fluoresced strongly by LFA. The results demonstrate that acute infection with Orientia tsutsugamushi results in a marked reduction of the HIV-1 viral load in some AIDS patients. Example 2 Anti-HIV activity of serum from scrub typhus patients Sera taken from scrub typhus patients who were HIV- 1 seronegative was tested for effect on HIV- 1 activity in vitro. Syncytia inhibition by mouse sera. Syncytia were counted 4 days after CEM- SS cells were infected with a laboratory-adapted clade E HIV-1 strain (NP03) at 1:20 or 1:80 dilution of test sera (18). Samples and preimmune controls were run in quadruplicate. Infectivity reduction assays (19). PHA-stimulated PBMCs from a US donor were inoculated for 1.5h with a primary, NSI HIV subtype E isolate (NP 1660). The inoculum was removed, and cells washed and incubated for 18-24h in media containing 10% interleukin-2. Test sera were then added at a dilution of 1 :20 with normal human sera (NHS) and sera from HIV positive donors as controls. Similar experiments were performed using H9 cells and NP03 virus before and after protein G antibody depletion. The % inhibition was 1 - (p24 test sample/ p24 NHS) X 100. Immunoassays (Table 3). Sera from 1 HIV-uninfected scrub typhus patient (coded KDX5) had neutralizing activity exceeding that of HIV positive control sera and was further characterized. KDX5 HIV-inhibition was markedly reduced after antibody depletion. There was IgG ELISA reactivity to pl7 recombinant protein in sera taken 14 days after scrub typhus infection, but not in day 450 sera. Day O and day 14 sera (but not day 28 or day 450 sera) had IgM reactivity to several pl7 epitopes by peptide scanning, including an epitope reactive with HIV positive serum. Convalescent sera from mice infected with scrub typhus (isolate SV240) inhibited HIV syncytia formation and was further tested. Day 28 SV240 serum (but not preimmune serum) reacted strongly with the p 17 band and weakly with the p55 band on IgM-specific Western blot and gave a fluorescent signal when incubated with HIV-infected lymphocytes (Figure 2). The results demonstrate that sera taken from HIV-1 seronegative patients during and after infection with scrub typhus shows anti-HIV- 1 activity in vitro. Example 3 Cross-reactivity of scrub typhus antibodies with HIV-1 pl7 gag protein IgG and IgM antibodies made during and after an acute scrub typhus infection were tested for cross-reactivity with HIV-1 using standard ELISA techniques. The results, shown in Figure 3, demonstrate that IgG and IgM antibodies made during acute scrub typhus infection are cross-reactive with HIV-1 pi 7 gag protein. Scrub typhus infection also causes mice to acquire strong antibodies to a gag pi 7 epitope, as shown in Figure 4. Reactivity to pi 7 peptides (pep 41 and pep 129) for mice infected with three different strains of scrub typhus is shown in Figure 5. Example 4 Sequence similarity between surface antigen oiOrientia tsutsugamushi and HIV-1 gag gene. The sequences of surface antigen of O. tsutsugamushi and HIV-1 gag gene were compared. The results are shown in Tables 5-7. Table 5 PROSIΞ ***** AMINO ACID MAXIMUM HOMOLOGY LIST ***** «INFORMATIONS EQUIVALENT Ala (A) , Ser(S) ,Thr(T) ,Pro(P) ,Gly(G) Asn(N), Asp(D), Asx(B) , Glu (E) , Gln(Q) , Glx(Z) His(H) ,Arg(R) ,Lys(K) Met(M), Leu(L) ,Ile(I) ,Val(V) Phe(F), Tyr (Y) ,Trp( ) FILE1 NAME START END FILE2 NAME START END PROT . MI 511 56KD.AMI 8 FILE NAME 1 10 20 30 40 50 60 PROT . AMI MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFAVNPGLLETSEGGRQI 56KD.AMI -184 -175 -165 -155 -145 -135 -125 FILE NAME 61 70 80 90 100 110 120 PROT .AMI LGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALDKIEEEQNKSKKKAQQAAA 56KD.AMI -124 -115 -105 -95 -85 -75 -65 FILE NAME 121 130 140 150 160 170 180 PROT . AMI DTGHSSQVSQNYPIVQNIQGQMVHQAISPRTLNA VKWEEKAFSPEVIPMFSALΞEGAT 56KD.AMI -64 -55 -45 -35 -25 -15 -5 FILE NAME 181 190 200 210 220 230 240 PQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDKVMPVMAGPIAPGQMREPRGΞDIAGTT 56KD.AMI .A--DSVGGKD. 5 15 25 35 45 55 FILE NAME 241 250 260 270 280 290 300 PRO .AMI STLQEQIG MTNNPPIPVGEIYKR IILGLNKIVRMYSPTSILDIRQGPKEPFRDYVRFY 56KD.AMI 56 65 75 85 95 105 115 FILE NAME 301 310 320 330 340 350 360 PROT .AMI KTLRAEQASQEVKN MTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKAR 56KD.AMI 116 125 135 145 155 165 175 FILE NAME 361 370 380 390 400 410 420 VLAEAMSQVTNSATIMMQRGNFRNQRKIVKCFNCGKEGHIARNCRAPRKKGC KCGKEGH 56KD.AMI 176 185 195 205 215 225 235 FILE NAME 421 430 440 450 460 470 480 PROT .AMI QMKDCTERQANFLGKIWPSYKGRPGNFLQSRPEPTAPPFLQSRPEPTAPPEESFRSGVET 56KD.AMI 236 245 255 265 275 285 295 FILE NAME 481 490 500 510 PROT .AMI TTPSQKQEPIDKELYPLTSLRSLFGNDPSSQ 56KD.AMI 296 305 315 325 MATCHING PERCENTAGE TOTAL WINDOW 1% 511) ALIGNMENT WINDOW 80% 10) Table 6 10 20 30 40 50 60 PROT.AMI MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFAVNPGLLETSEGGRQI STA56.AMI SAMSALSLPFSASAIELGEEGLECGPYAKVGWGGMITGVESARLDPADAEGKKHLSLTN 10 20 30 40 50 60 70 80 90 100 110 120 PRO .AMI LGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALDKIEEEQNKSKKKAQQAAA STA56.AMI GLPFGGTLAAGMTIAPGERAEIGVMYLTNITAQVEEGKVKADSVGETKADSVGGKDAPIR 70 80 90 100 110 120 130 140 150 160 170 180 PROT .AMI DTGHSSQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKWEEKAFSPEVIPMFSALSEGAT STA56.AMI KRFKLTPPQPTIMPISIAVRDFGIDIPNQTSAASTSRΞLRLNDEQRAAARIAWLKNCAGI 130 140 150 160 170 180 190 200 210 220 230 240 PROT . AMI PQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDKVMPVMAGPI PGQMREPRGSDI GTT STA56.AMI DYRVKNPNDPNGPMVINPILLNIPQGNPNPVGNPPQRANPPAGFAIHNHEQWRHLWGLA 190 200 210 220 230 240 250 260 270 280 290 300 PROT.AMI STLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVRFY v V Λ Λ STA56.AMI ALΞNANKPSASPVKVLSDKITQIYSDIKHLADIAGIDVPDTSLPNSASVEQIQNKMQELN 250 260 270 280 290 300 310 320 330 340 350 360 PROT .AMI KTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKAR STA56.AMI DLLEELRESFDGYLGGNAFANQIQLNFVMPQQAQQQGQGQQQQAQATAQEAVAAAAVRLL 310 320 330 340 350 360 370 380 390 400 410 420 PROT . AMI VLAEAMSQVTNSATIMMQRGNFRNQRKIVKCFNCGKEGHIARNCRAPRKKGCWKCGKEGH STA56.AMI NGNDQIAQLYKDLVKLQRHAGIKKAMEKLAAQQEEDAKNQGEGDCKQQQGTSEKSKKGKD 370 380 390 400 410 420 430 440 450 460 470 480 PROT . AMI QMKDCTERQANFLGKIWPSYKGRPGNFLQSRPEPTAPPFLQSRPEPTAPPEESFRSGVET STA56.AMI KEAEFDLSMIVGQVKLYADVMITESVSIYAGVGAGLAYTSGKIDNKDIKGHTGMVASGAL 430 440 450 460 470 480 490 500 510 PROT .AMI TTPSQKQEPIDKELYPLTSLRSLFGNDPSSQ STA56.AMI GVAINAAEGVYVDIEGSYMYSFSKIEEKYSI 490 500 510 Table 7 10 20 30 40 50 60 PROT . AMI MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFAVNPGLLETSEGGRQI STA56.AMI ADSVGGKDAPIRKRFKLTPPQPTIMPISIAVRDFGIDIPNQTSAASTSRSLRLNDEQRAA 120 130 140 150 160 170 70 80 90 100 110 120 PROT .AMI LGQLQPS QTGSEELRΞLYNTVATLYCVHQRIEIKDTKEALDKIEEEQNKSKKKAQQ AA STA56.AMI ARI WLKNCAGIDYRVKNPNDPNGPMVINPILLNIPQGNPNPVGNPPQRANPPAGFAIHN 180 190 200 210 220 230 130 140 150 160 170 180 PROT . AMI DTGHSSQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKWEEKAFSPEVIPMESALSEGAT STA56.AMI HEQWRHLWGLAALSNANKPSASPVKVLSDKITQIYSDIKHLADIAGIDVPDTSLPNSAS 240 250 260 270 280 290 190 200 210 220 230 PROT .AMI PQDLNTMLNTVGGHQAAM-QMLKETINEEAAEWDKVMPVMAGPIAPGOMREPRGSDIAGT : . . Λ . V . :. ..V. . :.. STA56.AMI VEQIQNKMQELNDLLEELRESFDGYLGGNAFANQIQLNFVMPQQAQQQGQGQQQQAQATA 300 310 320 330 340 350 240 250 260 270 280 290 PROT.AMI TSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVRF STA56.AMI QEAVAAAAVRLLNGND-QIAQLYKDLVKLQRHAGIKKAMEKLAAQQEEDAKNQGEGDCKQ 360 370 380 390 400 410 300 310 320 330 340 350 PROT.AMI YKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKA STA56.AMI QQGTSEKSKKGKDKEAEFDLSMIVGQVKLYADVMITESVSIYAGVGAGLAYTΞGKIDNKD 420 430 440 450 460 470 360 370 380 390 400 410 PROT .AMI RVLAEAMSQVTNSATIMMQRGNFRNQRKIVKCFNCGKEGHIARNCRAPRKKGCWKCGKE STA56.AMI IKGHTGMVAΞGALGVAINAAEGVYVDIEGSYMYSFSKIEEKYSINPLMASVSVRYNFQT 480 490 500 510 520 530 The results show that there are significant similarities between the amino acid sequences of an antigen on the surface of Orientia tsutsugamushi and sequences encoded by the HIV-1 gag gene. Example 5 Scrub typhus strains with anti-HTV activity can be identified as follows. 1. Isolation of Orientia tsutsugamushi from human blood 0.2 cc of whole blood from patients with acute scrub typhus is injected intraperitoneally (IP) into each of 3 mice using a sterile 26 gauge 1 inch needle. The needle is changed between mice and the abdomen cleansed with alcohol before each injection. Mice are checked daily for 14 days and euthanized if they show signs of illness. Spleens are harvested and divided into two sections. One half is placed in a vial and stored at -70°C, and the other is homogenized with 2.0 ml of Snyder's solution to make a 10-20% suspension. Two or more mice are injected IP with 0.2 ml of the suspension and observed for 28 days. If they become sick (e.g. rough coat, inactivity) within 28 days they are euthanized. Giemsa stained peritoneal scrapings are checked for the presence of O. tsutsugamushi and the spleens from infected mice stored at -70°C, as detailed by Watt et al. (20). Blood from about forty patients with scrub typhus should yield about 10 new isolates of the organism. 2. Immunoassays To generate immunological reagents, outbred ICR mice are infected with O. tsutsugamushi (1000 X mouse lethal doseso; intraperitoneally) using organisms isolated from patients with scrub typhus as previously described (20). Sera are obtained 28 days after infection by intracardiac puncture. Sera are assayed on Western blots of whole HTV-1 viral lysate protein (Novapath R HIV-1 Immunoblot, Bio-Rad, Hercules, California, USA) using standard secondary antibodies labelled with horse-radish peroxidase and an anti-mouse IgM heavy chain conjugate. Positive and negative controls supplied by the manufacturer and preimmune sera are used in comparisons. Detection of HP/ reactive antibodies is conducted using indirect immunofluorescence with H9 cells infected with HIV-1 (NP03) for 96h. Test sera are incubated with the cells at 37°C for 30min, washed and incubated with FITC-labelled anti-IgG conjugate. Positive control antibody is mouse monoclonal anti-gag antibody produced against HIV subtype B (NIH AIDS Research and Reference Reagent Program). Negative controls are uninfected H9 cells and pre-immune antisera. Human sera are assayed by ELISA for p 17 antibody using recombinant pl7 HIV-1 core protein (Immunodiagnostics Inc, Bedford, MA) at a coating concentration of 1 ug/ml. Positive controls are sera from HfV-infected subjects reactive with pl7 antigen by Western blot. Negative controls included sera from HIV-uninfected individuals and sera added to wells not coated with pi 7 antigen. Eight amino acid (aa) overlapping 12-mer peptides were synthesized according to the pl7 aa sequence of a Northern Thai HrV clade E isolate (CM243). For results presented herein, ELISA reactivities were assayed as previously described (21), except that an IgM-specific conjugate was used. Experiments included sera from HrV infected patients as well as HTV uninfected patients with acute scrub typhus. Acute phase reactants and immune activation. Serum C-reactive protein (CRP) concentration was measured by serial radial diffusion with a lower limit of detection of 3.3 mg/L; values above 5.0 mg/L were abnormal. TNF-alpha concentration was determined by ELISA using commercially available kits (R&D Systems, Minneapolis, Minn) with an assay range of 15.6-1,000 pg/ml. RANTES (normal range 24,230-85,990 pg/ml) and MIP-1 alpha (limit of detection 80 pg/ml) were assayed by ELISA using commercially available kits (R&D Systems) on the first five HIV seropositive patients infected with O. tsutsugamushi. Statistical Analyses Repeated measures were analyzed using multivariate analysis and the Linear General Hypothesis (SYSTAT v5.2, SYSTAT Corp. Evanston, IL). Proportions were compared with chi-square analysis or Fisher's exact test (StatView, SAS Institute Inc. Cary, NC). Values not normally distributed were analyzed by the Mann-Whitney U statistic or Kolmogorov- Smirnoff test as appropriate. Two-sided tests of significance were measured in all cases. Changes in viral loads were related to the measurement made 28 days after admission to the study in accordance with recommendations made by an NIH advisory panel (22). This time point most closely represents viral status after resolution of symptoms and signs of acute infection and completion of therapy. Raw viral load data were converted to percentages related to the day 28 value ( 100%) to adjust for inter-assay differences. Changes greater than threefold in either direction on repeated measurements are considered to reflect a biologically relevant change in the level of plasma HIV RNA (22). For peptide scanning, optical densities were expressed in standard deviations from the median reactivity to background peptides; reactivity more than 5 times that of background was considered significant. Useful variations of this method and other means for identifying such strains will be apparent to those of skill in the art. References cited herein are listed below for convenience and are hereby incorporated by reference. REFERENCES 1. Bloom, BR. The highest attainable standard: ethical issues in AIDS vaccines. Science 1998;279: 186-188. 2. Egger, M, Hirschel, B, Francioli, P, et al. Impact of new antiretroviral combination therapies in HIV infected patients in Switzerland: prospective multicentre study. Brit. Med J. 1997; 315: 1194-9. 3. Katz, MH, Gerberding, JL. The care of persons with recent sexual exposure to HIV. Ann Intern Med 1998;128:306-12. 4. Kantipong, P. Watt, G., Jongsakul, K, Chuenchitra, C. HIV infection does not influence the clinical severity of scrub typhus. Clin. Inf. Dis. 1996: 23: 1 168-70. 5. Watt G, Strickman D, Kantipong P, Jongsakul K, Paxton H. Prospective evaluation of a dotblot immunoassay for the rapid diagnosis of scrub typhus. J Inf Dis 1998; 177:800-2. 6. Winslow WE, Merry DJ, Pirc ML, Devine PL. Evaluation of a commercial enzyme- linked immunosorbent assay for detection of immunoglobulin M antibody in diagnosis of human leptospiral infection. J Clin Micro 1997; 35: 1938-42. 7. Artenstein AW, VanCott TC, Mascola J et al. Dual infection with human immunodeficiency virus type 1 of distinct envelope subtypes in humans. J Inf Dis 1995; 171:805-10. 8. Division of AIDS, National Institute of Alllergy and Infectious Diseases, 1997. DAIDS Virology Manual for HIV Laboratories. Publicaton NIH-97-3828. U.S. Department of Health and Human Services, Washington D.C. 9. McGuire W, D'Alessandro I, Olaley BO, Thomson MC, Langerock P, Greenwood BM, Kwiatowski D. C-reactive protein and haptoglobin in the evaluation of a community- based malaria control program. Trans Roy Soc Trop Med Hyg 1996; 90: 10-14. 10. Sulkowski MS, Chaisson RE, Karp CL et al. The effect of acute infectious illnesses on plasma human immunodeficiency virus (HIV) type 1 load and the expression of serologic markers of immune activation among HIV-infected adults. J Inf Dis 1998; 178:1642-8. 1 1. Mole L, Ripich S, Margolis D, Holiday M. The impact of active herpes simplex virus infection of human immunodeficiency virus load. J Inf Dis 1997; 176:766-70. 12. Brichacek B, Swindells, Janoff EN et al. Increased plasma human immunodeficiency virus type 1 burden following antigenic challenge with pneumococcal vaccine. J Inf Dis 1996; 174:1191-9. 13. Ortigao-de-Sampaio, Shattock RJ, Hayes P, Griffin GE, Linhares-de-Carvalho MI, Ponce de Leon A, Lewis DJM, Castello-Branco LRR. Increase in plasma viral load after oral cholera immunization of HIV-infected subjects. AIDS 1998; 12:F145-50. 14. Garcia F, Vidal G, Gatell JM et al. Changes in HIV-1 RNA viral load following tuberculin skin test. JAIDS 1998; 18:398-9. 15. Oaks EV, Rice RM, Kelly DJ, Stover CK. Antigenic and genetic relatedness of eight Rickettsia tsutsugamushi antigens. Inf Imm 1989; 57:3116-22. 16. Richmann DD, Bozzette SA. The impact of the syncytium-inducing phenotype of human immunodeficiency virus on disease progression. J Inf Dis 1994; 169:968-74. 17. Fais S, Capobianchi MR, Abbate I, Castilletti C, Gentile M, Fei PC, Ameglio F, Dianzani F. Unidirectional budding of HIV-1 at the site of cell to cell contact is associated with co- polarization of intercellular adhesion molecules and HIV-1 viral matrix protein. AIDS 1995; 9:329-35. 18. Nara PL, Fischinger PJ. Quantitative infectivity assay for HIV-1 and -2. Nature 1988; 332:70. 19. Zolla-Pazner S, Alving C, Belshe R et al. Neutralization of a clade B primary isolate by sera from human immunodeficiency virus-uninfected recipients of candidate AIDS vaccines. J Inf Dis 1997; 175:764-74. 20. Watt G, Chouriyagune C, Ruangweerayud R et al. Scrub typhus infections poorly responsive to antibiotics in Northern Thailand. Lancet 1996; 348:86-89. 21. Loomis-Price LD, Cox JH, Mascola JR et al. Correlation between humoral responses to human immunodeficiency virus type 1 envelope and disease progression in early-stage infection. J Inf Dis 1998; 178:1306-16. 22. Report of the NIH panel to define principles of therapy of HTV infection. Ann Int Med 1998, 128:1057-78.

Claims: WHAT IS CLAIMED IS: 1. Isolated and purified blood serum or plasma from a patient diagnosed as having been infected with scrub typhus, said serum having been identified as containing an inhibitory factor having anti-HIV activity. 2. The serum or plasma of claim 1 that contains anti-scrub typhus antibody that recognizes HTV p24 antigen and/or p 17 antigen. 3. An isolated suppressor factor derived from blood of a mammal infected with scrub typhus, said factor having anti-HIV activity. 4. The factor of claim 3 wherein said mammal is a mouse. 5. The factor of claim 3 wherein said mammal is a human. 6. Isolated scrub typhus antibodies which cross-react with HIV-1 proteins. 7. Antibodies of claim 6 which neutralize HIV-1 virus. 8. A factor derived from scrub typhus infection which neutralizes HIV-1 virus infectivity. 9. A factor derived from scrub typhus infection which suppresses HF -1 virus replication. 10. A vaccine for passive immunization against HIV infection comprising an antibody of claim 2. 11. A method for prevention or treatment of HIV infection comprising administration of the vaccine of claim 10. 12. A vaccine for passive immunization against HIV infection comprising an antibody of claim 7. 13. A method for prevention or treatment of HIV infection comprising administration of the vaccine of claim 12. 14. An immunogenic composition for active immunization against HTv * infection comprising a peptide or peptides from O. tsutsugamushi which induce an immunogenic response directed against HΓV-1. 15. The peptide of claim 14 which is set forth in SEQ ID NO:2 or SEQ ID NO:4 or a fragment thereof. 16. A method of inducing an immunogenic response to HTV infection in an individual comprising administration of an effective amount of the immunogenic composition of claim 14. 17. A method for prevention or treatment of HIV infection comprising administration of the serum or plasma of claim 1. 18. A method for prevention or treatment of HIV infection comprising administration of the serum or plasma of claim 2.

Inventors: BIRX DEBORAH L, BROWN ARTHUR E, DESOUZA MARK S, SIRIYAPHAN PHINYO, WATT GEORGE H, BIRX, DEBORAH, L., BROWN, ARTHUR, E., DESOUZA, MARK, S., SIRIYAPHAN, PHINYO, WATT, GEORGE, H.
Applicants: BIRX DEBORAH L, BROWN ARTHUR E, DESOUZA MARK S, SIRIYAPHAN PHINYO, WATT GEORGE H, BIRX, DEBORAH, L., BROWN, ARTHUR, E., DESOUZA, MARK, S., SIRIYAPHAN, PHINYO, WATT, GEORGE, H.
Application year: 1999
Main classification: C07K16
IPC codes: A61K38, A61K39, C07K14, C07K16