The rationale of our research program rests on the following three points.
1. Next-generation influenza vaccines will require immunogens that elicit humoral responses sufficiently broad to prohibit escape along ordinary pathways of viral evolution (antigenic drift). A second goal will be immunogens that generate pre-pandemic immunity (anticipate a likely antigenic shift).
2. A vaccine with these properties would elicit a broad, polyclonal response to multiple evolutionarily constrained epitopes on the viral hemagglutinin (HA) and neuraminidase (NA).
3. To propose and test a convincing route to rational design of such a vaccine, the influenza vaccine research community needs: (a) a deeper understanding of human B-cell responses to HA and NA and of the influence of T-cell epitope diversity on humoral responses; (b) a more complete description of B-cell dynamics in immune imprinting by primary exposure and recall upon secondary exposure, leading to updating by germinal center re-entry and further affinity maturation; (c) a picture of how updating of humoral memory relates to the molecular characteristics of the primary and secondary immunogens and to antigenic differences between them; (d) an assessment of the relative importance of memory B cells and long-lived plasma cells for durable protection.
Aim 1. Imprinting, affinity maturation, and evolution of the B cell response to HA and NA
Hypotheses: We can infer how imprinting by different initial exposures affects later responses and assess the limits of affinity maturation, by studying, in individual human subjects from different age groups, their BCR and serum Ab repertoires in response to the same vaccine.
Aim: Analyze accessible BCR repertoire components -- plasma cells, circulating Bmem, and serum antibodies -- in individuals of different ages who have received the same vaccine: seniors (age >65); adults (18-65, stratified by birth year); teens (12-18) in 2 categories -- vaccinated as infants or not; and infants after their first influenza immunization and after year 2 boost.
Aim 2. Beyond antibody specificity: influence of locality
Hypothesis 1: Humoral immune memory encompasses specificity, affinity, and location. Responses to protein immunogens “remember” immunization or infection sites by recruiting (after local boost) local Bmem into secondary GCs; distal-site boosts, in contrast, elicit equally robust GC responses but with little or no Bmem participation. GC-resident, follicular helper T cell (TFH) distributions and participation in boost responses also depend on location.
Aim: B-cell memory in mice. Characterize BCR specificity, affinity and genetics of GC B cells, Bmem, and PCs, after primary immunizations of mice in which GC B cells and their progeny have been conditionally marked; determine participation of marked B cells in secondary GC responses after boosts at the same or distal sites. Extend study to intranasal infections or intranasal or subcutaneous immunizations, followed by intranasal challenge with homologous or heterologous viruses.
Aim: B-cell memory in non-human primates. Quantify and characterize serum IgG antibody responses and determine BCR repertoires in GC, Bmem, and PC compartments from blood, draining lymph nodes, bone marrow, and respiratory sites.
Aim: TFH distributions in mice. Characterize the numbers, distribution, and TCRβ repertoire of primary TFH cells after immunizations of mice in which GC TFH and their progeny have been conditionally marked. Determine the participation of marked TFH in secondary responses at the same and distal sites.
Hypothesis 2: We can assess the relative importance of Bmem and LLPC in protective responses to homologous and heterologous virus by performing conditional deletion experiments in mice.
Aim: Immunize, with HA or NA protein, mice that conditionally express the diphtheria toxin receptor on Bmem, but not LLPC, in order to isolate the roles of Bmem and LLPC in resistance to subsequent intranasal challenge with homologous or heterologous viral strains.
Aim 3. Immunogen design and viral evolution
Hypothesis: Epitope "grafting" (resurfacing) can be used to probe underlying features of the B cell immune response to HA and NA; grafted immunogens can test fundamental immune response properties.
Aim: Use HA trimeric chimeras to probe use of broadened T-cell help for immunogen design
Aim: Adapt to NA the epitope grafting and hyperglycosylation strategies we have used with HA and use these immunogens to manipulate the NA immune response. Are the "rules" of imprinting and recall similar to those for HA? Which epitopes are recognized by antibodies that confer some degree of protection?
Aim: Study virus antibody co-evolution by in vitro selection. Can a particular sequence of immunizations exhaust the possibilities for escape at conserved epitopes and drive the virus into an evolutionary trap?