Suzanne L. Epstein

Prior H1N1 Influenza Infection and Susceptibility of Cleveland Family Study Participants during the H2N2 Pandemic of 1957: An Experiment of Nature

During a pandemic, influenza vaccines that rely on neutralizing antibodies to protect against matched viruses might not be available early enough. Much broader (heterosubtypic) immune protection is seen in animals. Do humans also have cross-subtype immunity? To investigate this issue, archival records from the Cleveland Family Study, which was conducted before and during the 1957 pandemic (during which a shift from subtype H1N1 to H2N2 occurred), were analyzed. Only 5.6% of the adults who had had symptomatic influenza A in earlier study years developed influenza during the pandemic, despite living in households with participants who had influenza. In contrast, 55.2% of the children who had had symptomatic influenza A contracted it again. These findings suggest an impact of accumulated heterosubtypic immunity during a pandemic. Such immunity, as well as its implications for vaccination, should be further investigated.

Matrix Protein 2 Vaccination and Protection against Influenza Viruses, Including Subtype H5N1

Vaccination of mice with influenza matrix protein 2 induced cross-reactive antibody responses.

Critical Roles for the Bcl-3 Oncoprotein in T Cell–Mediated Immunity, Splenic Microarchitecture, and Germinal Center Reactions

Chromosomal translocations of bcl-3 are associated with chronic B cell lymphocytic leukemias. Previously, we have shown that Bcl-3, a distinct member of the I kappa B family, may function as a positive regulator of NF-kappa B activity, although its physiologic roles remained unknown. To uncover these roles, we generated Bcl-3-deficient mice. Mutant mice, but not their littermate controls, succumb to T. gondii owing to failure to mount a protective T helper 1 immune response. Bcl-3-deficient mice are also impaired in germinal center reactions and T-dependent antibody responses to influenza virus. The results reveal critical roles for Bcl-3 in antigen-specific priming of T and B cells. Altered microarchitecture of secondary lymphoid organs in mutant mice, including partial loss of B cells, may underlie the immunologic defects. The implied role of Bcl-3 in maintaining B cells in wild-type mice may related to its oncogenic potential.

Heterosubtypic Immunity to Influenza A Virus in Mice Lacking IgA, All Ig, NKT Cells, or γδ T Cells

The mechanisms of broad cross-protection to influenza viruses of different subtypes, termed heterosubtypic immunity, remain incompletely understood. We used knockout mouse strains to examine the potential for heterosubtypic immunity in mice lacking IgA, all Ig and B cells, NKT cells (CD1 knockout mice), or γδ T cells. Mice were immunized with live influenza A virus and compared with controls immunized with unrelated influenza B virus. IgA−/− mice survived full respiratory tract challenge with heterosubtypic virus that was lethal to controls. IgA−/− mice also cleared virus from the nasopharynx and lungs following heterosubtypic challenge limited to the upper respiratory tract, where IgA has been shown to play an important role. Ig−/− mice controlled the replication of heterosubtypic challenge virus in the lungs. Acute depletion of CD4+ or CD8+ T cell subsets abrogated this clearance of virus, thus indicating that both CD4+ and CD8+ T cells are required for protection in the absence of Ig. These results in Ig−/− mice indicate that CD4+ T cells can function by mechanisms other than providing help to B cells for the generation of Abs. Like wild-type mice, CD1−/− mice and γδ−/− mice survived lethal heterosubtypic challenge. Acute depletion of CD4+ and CD8+ cells abrogated heterosubtypic protection in γδ−/− mice, but not B6 controls, suggesting a contribution of γδ T cells. Our results demonstrate that the Ab and cellular subsets deficient in these knockout mice are not required for heterosubtypic protection, but each may play a role in a multifaceted response that as a whole is more effective than any of its parts.

TLR3-Specific Double-Stranded RNA Oligonucleotide Adjuvants Induce Dendritic Cell Cross-Presentation, CTL Responses, and Antiviral Protection

Maturation of dendritic cells (DC) to competent APC is essential for the generation of acquired immunity and is a major function of adjuvants. dsRNA, a molecular signature of viral infection, drives DC maturation by activating TLR3, but the size of dsRNA required to activate DC and the expression patterns of TLR3 protein in DC subsets have not been established. In this article, we show that cross-priming CD8α+ and CD103+ DC subsets express much greater levels of TLR3 than other DC. In resting DC, TLR3 is located in early endosomes and other intracellular compartments but migrates to LAMP1+ endosomes on stimulation with a TLR3 ligand. Using homogeneous dsRNA oligonucleotides (ONs) ranging in length from 25 to 540 bp, we observed that a minimum length of ∼90 bp was sufficient to induce CD86, IL-12p40, IFN-β, TNF-α, and IL-6 expression, and to mature DC into APC that cross-presented exogenous Ags to CD8+ T cells. TLR3 was essential for activation of DC by dsRNA ONs, and the potency of activation increased with dsRNA length and varied between DC subsets. In vivo, dsRNA ONs, in a size-dependent manner, served as adjuvants for the generation of Ag-specific CTL and for inducing protection against lethal challenge with influenza virus when given with influenza nucleoprotein as an immunogen. These results provide the basis for the development of TLR3-specific adjuvants capable of inducing immune responses tailored for viral pathogens.

Vaccination focusing immunity on conserved antigens protects mice and ferrets against virulent H1N1 and H5N1 influenza A viruses.

Immunization against conserved virus components induces broad, heterosubtypic protection against diverse influenza A viruses, providing a strategy for controlling unexpected outbreaks or pandemics until strain-matched vaccines become available. This study characterized immunization to nucleoprotein (NP) and matrix 2 (M2) by DNA priming followed by parenteral or mucosal boosting in mice and ferrets. DNA vaccination followed by boosting with antigen-matched recombinant adenovirus (rAd) or cold-adapted (ca) influenza virus provided robust protection against virulent H1N1 and H5N1 challenges. Compared to other boosts, mucosal rAd induced stronger IgA responses, more virus-specific activated T-cells in the lung, and better protection against morbidity following challenge even eight months post-boost. In ferrets, both mucosal and parenteral rAd boosting protected from lethal H5N1 challenge. These findings demonstrate potent protection by vaccination highly focused on conserved antigens and identify immune response measures in mice that differed among vaccinations and correlated with outcome.

Single-Dose Mucosal Immunization with a Candidate Universal Influenza Vaccine Provides Rapid Protection from Virulent H5N1, H3N2 and H1N1 Viruses

Background The sudden emergence of novel influenza viruses is a global public health concern. Conventional influenza vaccines targeting the highly variable surface glycoproteins hemagglutinin and neuraminidase must antigenically match the emerging strain to be effective. In contrast, “universal” vaccines targeting conserved viral components could be used regardless of viral strain or subtype. Previous approaches to universal vaccination have required protracted multi-dose immunizations. Here we evaluate a single dose universal vaccine strategy using recombinant adenoviruses (rAd) expressing the conserved influenza virus antigens matrix 2 and nucleoprotein. Methodology/Principal Findings In BALB/c mice, administration of rAd via the intranasal route was superior to intramuscular immunization for induction of mucosal responses and for protection against highly virulent H1N1, H3N2, or H5N1 influenza virus challenge. Mucosally vaccinated mice not only survived, but had little morbidity and reduced lung virus titers. Protection was observed as early as 2 weeks post-immunization, and lasted at least 10 months, as did antibodies and lung T cells with activated phenotypes. Virus-specific IgA correlated with but was not essential for protection, as demonstrated in studies with IgA-deficient animals. Conclusion/Significance Mucosal administration of NP and M2-expressing rAd vectors provided rapid and lasting protection from influenza viruses in a subtype-independent manner. Such vaccines could be used in the interval between emergence of a new virus strain and availability of strain-matched vaccines against it. This strikingly effective single-dose vaccination thus represents a candidate off-the-shelf vaccine for emergency use during an influenza pandemic.

Cross-protective immunity to influenza A viruses

Antigenic changes in influenza virus occur gradually, owing to mutations (antigenic drift), and abruptly, owing to reassortment among subtypes (antigenic shift). Availability of strain-matched vaccines often lags behind these changes, resulting in a shortfall in public health. In animal models, cross-protection by vaccines based on conserved antigens does not completely prevent infection, but greatly reduces morbidity, mortality, virus replication and, thus, viral shedding and spread. Such immunity is especially effective and long-lasting with mucosal administration. Cross-protective immunity in humans is controversial, but is suggested by some epidemiological findings. ‘Universal’ vaccines protective against all influenza A viruses might substantially reduce severity of infection and limit spread of disease during outbreaks. These vaccines could be used ‘off the shelf’ early in an outbreak or pandemic, before strain-matched vaccines are available.

Comparison of vaccines for induction of heterosubtypic immunity to influenza A virus: Cold-adapted vaccine versus DNA prime-adenovirus boost strategies

Influenza epidemics or pandemics can arise for which strain- or subtype-matched vaccines are unavailable. Heterosubtypic immunity (Het-I) targeting conserved influenza A antigens could reduce morbidity and mortality during preparation of matched vaccines. Various vaccines inducing Het-I in animals have been studied separately using different viruses and conditions, but effectiveness for inducing Het-I has not been directly compared. The present studies compared immunization with cold-adapted (ca) viruses to DNA prime-recombinant adenovirus (rAd) boost vaccination to conserved antigens nucleoprotein (NP), matrix-2 (M2), or A/NP+M2. Both ca and DNA-rAd vaccinations induced antibody and T cell responses, and protected against lethal H1N1 challenge. Only A/NP+M2 DNA-rAd protected against challenge with highly pathogenic A/Vietnam/1203/2004 (H5N1); ca vaccine did not. Existing ca vaccines may provide some Het-I, but experimental vaccination focusing on conserved antigens was more effective in this model for protection against a divergent, highly pathogenic virus.

Cold-Adapted Influenza and Recombinant Adenovirus Vaccines Induce Cross-Protective Immunity against pH1N1 Challenge in Mice

Background The rapid spread of the 2009 H1N1 pandemic influenza virus (pH1N1) highlighted problems associated with relying on strain-matched vaccines. A lengthy process of strain identification, manufacture, and testing is required for current strain-matched vaccines and delays vaccine availability. Vaccines inducing immunity to conserved viral proteins could be manufactured and tested in advance and provide cross-protection against novel influenza viruses until strain-matched vaccines became available. Here we test two prototype vaccines for cross-protection against the recent pandemic virus. Methodology/Principal Findings BALB/c and C57BL/6 mice were intranasally immunized with a single dose of cold-adapted (ca) influenza viruses from 1977 or recombinant adenoviruses (rAd) expressing 1934 nucleoprotein (NP) and consensus matrix 2 (M2) (NP+M2-rAd). Antibodies against the M2 ectodomain (M2e) were seen in NP+M2-rAd immunized BALB/c but not C57BL/6 mice, and cross-reacted with pH1N1 M2e. The ca-immunized mice did not develop antibodies against M2e. Despite sequence differences between vaccine and challenge virus NP and M2e epitopes, extensive cross-reactivity of lung T cells with pH1N1 peptides was detected following immunization. Both ca and NP+M2-rAd immunization protected BALB/c and C57BL/6 mice against challenge with a mouse-adapted pH1N1 virus. Conclusion/Significance Cross-protective vaccines such as NP+M2-rAd and ca virus are effective against pH1N1 challenge within 3 weeks of immunization. Protection was not dependent on recognition of the highly variable external viral proteins and could be achieved with a single vaccine dose. The rAd vaccine was superior to the ca vaccine by certain measures, justifying continued investigation of this experimental vaccine even though ca vaccine is already available. This study highlights the potential for cross-protective vaccines as a public health option early in an influenza pandemic.