We summarize our overall rationale as follows:

1. Next-generation influenza vaccines will require immunogens that elicit humoral responses sufficiently broad to impair escape along ordinary pathways of viral evolution (antigenic drift).  An influenza vaccine that can generate pre-pandemic immunity (i.e., anticipate a likely antigenic shift)


2. A vaccine with the next-generation properties just described would probably need to elicit a broad 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, we need to answer to the following questions:

(a) What is the epitopic distribution of human B-cell responses to HA and NA?

(b) What are the repertoires of Bmem and LLPCs established by primary responses and what are the "rules" of Bmem re-activation and germinal-center (GC) re-entry vs. plasmablast differentiation in secondary responses? What is the relationship between the locations of primary infection or vaccination and secondary challenge or boost?

(c) How does updating of humoral memory (that is, the generation of cross-reactive Bmem or LLPCs in secondary GCs by affinity maturation to an antigenic variant such as an antigenically drifted or shifted HA or NA) relate to the molecular characteristics of the immunogen?

(d) What is the relative importance of Bmem and LLPCs for durable protection.

(e) How do the T-cell epitopes in an immunogen influence the outcome of the humoral response?  In particular, does T-cell epitope conservation contribute to immune imprinting?  How does T-cell epitope diversity contribute to strength of the B-cell response.

Aim 1.  Imprinting and updating

Hypotheses:  Global analysis of antibodies (Abs) in samples from human B-cell repertoires will probe the "rules" that determine B-cell fate decisions; differences among age groups imprinted by infection with different subtypes or by multivalent vaccination will reveal how imprinting affects immunodominance in vaccine responses and influences immunity to future pandemic viruses. 


Aim 1.1. Determine the distributions in various B-cell compartments (memory B cells, plasmablast burst following vaccination, long-lived plasma cells as reflected in serum Abs) of the specificities, epitopes recognized, affinities, and extents of SHM, for representatives of the full spectrum of age groups in our cohort.

Aim 1.2. Mimic some of the histories of human exposures in controlled experiments in non-human primates (NHPs), with analyses that include obtaining B cells from both lymph-node samples and blood.

Aim 2.  Beyond B-cell epitopes: locality of B-cell and TFH-cell memory; roles of long-lived plasmacytes and memory B cells in protection; contribution of T-cell epitopes to imprinting

Hypothesis: Vaccine efficacy depends on anatomical locations of prime and boost and on T-cell epitope conservation across distant strains. 


Aim 2.1. Test whether we observe in NHPs the same influence of relative location of prime and boost on recall vs. naive response that we have seen in mice.

Aim 2.2. Determine the role of localization for TFH-cell memory in mice. Determine whether the role of localization is as prominent for responses to non-lethal infection as it is to responses to protein immunization.

Aim 2.3. Use conditional deletion in mice to determine the relative roles of long-lived plasmacytes and memory B cells in protection from challenge.

Aim 2.4. Use designed immunogens to test whether conservation of T-cell epitopes contributes to immune imprinting.

Aim 3. Immunogen design and virus-antibody co-evolution

Hypotheses: (1) Appending or modulating T-cell epitopes on HA can alter the robustness of the elicited response while maintaining immune focusing properties. (2) Immunogen design strategies implemented for HA-based immune focusing to the receptor binding site can be extended to NA to elicit protective responses. (3) Coupling directed evolution platforms with in vitro viral resistance selection will aid in optimizing HA-based immunogens and determine whether multiple co-evolutionary cycles can lead to “exhaustion” of escape alternatives at a conserved viral epitope.


Aim 3.1.  Modulate T-cell epitopes on designed HA immunogens to test by immunization in mice whether T-cell epitope diversity contributes to the observed responses. 

Aim 3.2. Carry out cycles of NA immunogen design, tests in mice, and analyses of the serum response and of the specificities, epitopes recognized, and affinities of the memory BCRs.

Aim  3.3. Produce a "molecular movie" of virus-antibody co-evolution by coupling directed evolution of antibodies and selection for viral resistance