+---------+------------------------------------------------------------+
| **Abst  | Decline in immune function is a hallmark of aging, leading |
| ract:** | to increased susceptibility and severity of bacterial and  |
|         | viral infections in the elderly population. It is well     |
|         | documented that both innate and adaptive immune functions  |
|         | decline with aging leading to poor responses to preventive |
|         | vaccines. Vaccine strategies to improve immunity to both   |
|         | epidemic and pandemic influenza vaccines are urgently      |
|         | needed for this target population. We propose two novel    |
|         | vaccine strategies that could restore antigen presenting   |
|         | cell (APC) function and induce protective immune responses |
|         | to vaccines against H5N1 and H7N7avian influenza viruses   |
|         | in a preclinical aged mouse model. These studies address   |
|         | the NVPO priorities of adult immunization, pandemic        |
|         | preparedness and future vaccines.                          |
+---------+------------------------------------------------------------+
| **S     | Aim 1. Establish an H7 mouse model for evaluation of       |
| pecific | candidate H7 vaccines                                      |
| aims:** |                                                            |
|         | Aim 2: Evaluate a protein based H5, and H7 vaccine         |
|         | strategy that involves mobilizing dendritic cells (DCs)    |
|         | prior to vaccination in an influenza-primed aged mouse     |
|         | model                                                      |
|         |                                                            |
|         | Aim 3 Assess the immunogenicity and protective efficacy of |
|         | H5 and H7 HA DNA administered together with murine TLR5    |
|         | and its ligand, bacterial flagellin from *S.*              |
|         | *typhimurium* expressed under promoters that target        |
|         | macrophages or DCs.                                        |
+---------+------------------------------------------------------------+
| *       | The population of elderly in the world, including the      |
| *Backgr | United States, has increased considerably during the last  |
| ound:** | century. It is estimated that while the total U.S          |
|         | population will increase by 8% between 1991 and 2031, the  |
|         | elderly population between the ages of 60-84 will increase |
|         | by 48% and the population of persons over 85 years of age  |
|         | will increase by 128%. With aging, the ability to resist   |
|         | infectious diseases and respond to preventive vaccinations |
|         | decreases significantly leading to an increased incidence  |
|         | and severity of bacterial and viral infections of lungs,   |
|         | urinary tract, skin and soft tissues, which can result in  |
|         | death. For example, an estimated 90% of the 36,000 deaths  |
|         | on average that are attributed to influenza annually in    |
|         | the United States occur in persons aged 65 years.         |
|         | Although, the elderly are a major target group for annual  |
|         | influenza vaccination, the vaccine is only 30-40%          |
|         | effective in preventing influenza-like illness in frail    |
|         | elderly individuals, compared with 70-90% efficacy in      |
|         | preventing illness in healthy younger adults. A number of  |
|         | adaptive immune functions including cytokine secretion,    |
|         | clonal expansion and function of antigen-specific T and B  |
|         | cells, antigen presenting cell numbers and function        |
|         | decline or are altered with age, and are likely a major    |
|         | reason for the decreased efficacy of preventive            |
|         | vaccination in this age group. Recently, our laboratory    |
|         | provided the first evidence that the expression and        |
|         | function of Toll-like receptors (TLRs), pathogen sensors   |
|         | that provide defense against bacterial, mycotic and viral  |
|         | infections on macrophages, decline with aging. TLRs are    |
|         | also expressed on epithelial cells, dendritic cells and    |
|         | neutrophils. Since TLR expression and function is key to   |
|         | the initiation of an inflammatory response and induction   |
|         | of adaptive immunity, reduced expression of TLRs could     |
|         | explain why there is an increased severity of infectious   |
|         | diseases and poor responses to vaccination in the elderly. |
|         |                                                            |
|         | Since the currently licensed inactivated vaccine is        |
|         | suboptimal in protecting elderly against epidemic          |
|         | influenza, improved vaccine strategies are urgently needed |
|         | to protect this high risk population from severe or fatal  |
|         | illness. In addition, the elderly also represent a growing |
|         | population highly vulnerable to the next pandemic of       |
|         | influenza. The reemergence of highly pathogenic avian H5N1 |
|         | viruses in humans in Hong Kong in 2003 has renewed the     |
|         | status of this subtype as the top priority for pandemic    |
|         | vaccine development. H7N7 vaccines have also become a      |
|         | heightened priority for all ages, following the outbreak   |
|         | of highly pathogenic H7N7 viruses in Europe early in 2003, |
|         | with over 80 culture-confirmed human cases and some        |
|         | evidence of person-to-person transmission. Our goal is to  |
|         | evaluate in preclinical studies novel vaccine strategies   |
|         | that could overcome limitations in the aged immune         |
|         | response as well as intrinsic poor immunogenicity of avian |
|         | subtypes. We will use intradermal (gene gun) delivery of   |
|         | vaccine to express novel immunomodulators that may enhance |
|         | protective humoral as well as cell-mediated immune         |
|         | responses to avian influenza vaccines. Our goal is to      |
|         | identify strategies that will ensure optimal               |
|         | immunogenicity of both epidemic and pandemic influenza     |
|         | vaccines in elderly populations Thus these studies address |
|         | the NVPO high priority areas of adult immunization,        |
|         | influenza pandemic preparedness and future vaccines.       |
+---------+------------------------------------------------------------+

+---+--------------------------------------+----------------------------+
| * | **Aim 1: To develop a murine H7      |                            |
| * | model for vaccine evaluation** No    |                            |
| S | information currently exists on the  |                            |
| t | immunogenicity of H7 vaccines in     |                            |
| u | mammalian systems. Our previous work |                            |
| d | evaluating candidate H5 and H9       |                            |
| y | inactivated vaccines in murine       |                            |
| d | models correctly predicted the poor  |                            |
| e | immunogenicity of H5 vaccines in     |                            |
| s | humans and better immunogenicity of  |                            |
| i | an H9 vaccine We expect an H7 murine |                            |
| g | model to be equally useful. We will  |                            |
| n | investigate the pathogenicity of     |                            |
| / | H7N7 viruses from the recent         |                            |
| P | outbreak in the Netherlands and      |                            |
| l | other related H7 viruses and         |                            |
| a | establish the parameters upon which  |                            |
| n | vaccine efficacy can be determined.  |                            |
| : | Mice infected with various doses     |                            |
| * | (10^6^ to 10^1^ 50% egg infectious   |                            |
| * | doses (EID~50~) will be assessed for |                            |
|   | weight loss, death and viral         |                            |
|   | replication in respiratory and       |                            |
|   | systemic tissues. The optimal dose   |                            |
|   | for the induction of the disease     |                            |
|   | state in aged mice will be           |                            |
|   | determined.                          |                            |
|   |                                      |                            |
|   | **Aim 2**: **To assess the           |                            |
|   | immunogenicity and efficacy of       |                            |
|   | protein based H5 and H7 HA vaccines  |                            |
|   | following mobilization of dendritic  |                            |
|   | cells (DCs) prior to vaccination in  |                            |
|   | naïve and primed aged mice.**        |                            |
|   | Adaptive immune responses in aged    |                            |
|   | individuals may be compromised by    |                            |
|   | the lack of mobilization and         |                            |
|   | maturation of antigen presenting     |                            |
|   | cells such as dendritic cells (DCs)  |                            |
|   | and macrophages. We will attempt to  |                            |
|   | overcome this shortcoming by in vivo |                            |
|   | treatment with cytokines that        |                            |
|   | promote APC maturation and activity  |                            |
|   | prior to vaccination. To simulate    |                            |
|   | elderly human population with        |                            |
|   | respect to influenza exposure, we    |                            |
|   | will use antigen experienced         |                            |
|   | (infected with H1N1 or H3N2 viruses  |                            |
|   | while they were young) aged mice for |                            |
|   | all the proposed studies. We will    |                            |
|   | test purified baculovirus            |                            |
|   | recombinant H5, H7 and H9 HA         |                            |
|   | vaccines in the aged mice. Three     |                            |
|   | days prior to the administration of  |                            |
|   | vaccine, some groups of mice will    |                            |
|   | receive cytokines GMCSF or Flt3      |                            |
|   | ligand, either as protein or cDNA,   |                            |
|   | to facilitate differentiation and    |                            |
|   | mobilization of macrophages and DCs  |                            |
|   | respectively. We will administer one |                            |
|   | or two doses of vaccine by the       |                            |
|   | intramuscular and/or intradermal     |                            |
|   | route and evaluate the               |                            |
|   | immunogenicity and protective        |                            |
|   | efficacy of vaccination using the    |                            |
|   | following criteria:                  |                            |
|   |                                      |                            |
|   | -   Establish the kinetics of DC and |                            |
|   |     macrophage differentiation and   |                            |
|   |     mobilization following the       |                            |
|   |     administration of                |                            |
|   |     immunomodulatory cytokines by    |                            |
|   |     FACS analysis of draining        |                            |
|   |     lymphnodes;                      |                            |
|   |                                      |                            |
|   | -   Hemagglutination-inhibition      |                            |
|   |     (HI), neutralizing or IgG, IgM   |                            |
|   |     and IgA H5, H7 and H9            |                            |
|   |     HA-specific antibody responses;  |                            |
|   |                                      |                            |
|   | ```{=html}                           |                            |
|   | <!-- -->                             |                            |
|   | ```                                  |                            |
|   | -   Cytokine responses by CD4        |                            |
|   |     T-cells (IL-2, IFN-gamma, IL-4,  |                            |
|   |     and IL-5) following              |                            |
|   |     restimulation of spleen cells    |                            |
|   |     with appropriate avian HAs by    |                            |
|   |     the Bioplex cytokine assay;      |                            |
|   |                                      |                            |
|   | -   The frequency of                 |                            |
|   |     antigen-specific CD4 T-cells by  |                            |
|   |     ELISPOT or intracellular         |                            |
|   |     cytokine staining;               |                            |
|   |                                      |                            |
|   | -   The protective efficacy of the   |                            |
|   |     vaccines will be tested by       |                            |
|   |     challenge with the pathogenic    |                            |
|   |     H5N1, and H7N7 avian viruses.    |                            |
|   |     Weight loss, survival and viral  |                            |
|   |     tissue titers will be            |                            |
|   |     determined.                      |                            |
|   |                                      |                            |
|   | **Aim 3: Immunogenicity and          |                            |
|   | protection against homologous        |                            |
|   | challenge of H5, H7 and H9 HA DNA    |                            |
|   | based on expression of murine TLR5   |                            |
|   | and its ligand, bacterial flagellin  |                            |
|   | from *S.* *typhimurium*** Our lab    |                            |
|   | has shown that all known TLR         |                            |
|   | expression and function decline with |                            |
|   | aging in an aged mouse model. We     |                            |
|   | propose a novel way to express both  |                            |
|   | TLR as well as ligand to             |                            |
|   | reconstitute innate immune function. |                            |
|   | We will utilize DNA vaccine vectors  |                            |
|   | expressing a membrane-bound mTLR5,   |                            |
|   | secreted form of flagellin (fliC)    |                            |
|   | from S. typhimurium (as majority of  |                            |
|   | the ligands for various other TLRs   |                            |
|   | are not proteins) and membrane bound |                            |
|   | form of H5, H7 or H9 HA. These will  |                            |
|   | be expressed under CD11c or CD11b    |                            |
|   | promoters so that the expression is  |                            |
|   | restricted to Dendritic cells or     |                            |
|   | macrophages respectively. We         |                            |
|   | anticipate that the TLR 5 and its    |                            |
|   | ligand in DC and macrophages         |                            |
|   | initiate a robust inflammatory       |                            |
|   | response, which in turn will         |                            |
|   | generate a robust immune response to |                            |
|   | avian HA. We will immunize influenza |                            |
|   | H1N1 or H3N2 virus-primed aged mice  |                            |
|   | with the above constructs with gene  |                            |
|   | gun and examine both immunogenicity  |                            |
|   | and protection as detailed under     |                            |
|   | Specific Aim 1.                      |                            |
+---+--------------------------------------+----------------------------+