Respiratory syncytial virus (RSV) remains a major cause of morbidity and mortality, particularly among infants, the elderly, and individuals with high-risk conditions associated with severe outcomes. While neutralizing antibodies play a key role in protecting against initial infection and limiting viral replication, T cells are increasingly recognized as a critical component of the immune response to mitigate disease severity. Given the age-associated decline in T-cell function, evaluating T-cell responses to the mRNA-based RSV vaccine mRNA-1345 across age groups is important. In this clinical study (NCT05397223), healthy adults 18 to 75 years of age received 1 dose of mRNA-1345 (50 µg). Participant T-cell responses were evaluated by intracellular cytokine staining, activation-induced marker, and Meso Scale Discovery assays at several time points, up to 24 months after vaccination. Additionally, prefusion F (preF)-specific CD4+ and CD8+ T cells were sorted from peripheral blood mononuclear cells at baseline and 2 weeks after vaccination for single cell multiomic analysis. mRNA-1345 induced robust CD4⁺ T-cell responses in both younger and older adults, with responses sustained at 24 months postvaccination. Vaccination with mRNA-1345 also elicited an increase in CD8⁺ T-cell frequencies. The CD4⁺ T-cell response was strongly Th1-polarized, characterized by elevated IFN-γ, IL-2, and TNF-α production, with minimal Th2 cytokine expression, indicating a favorable immune profile for viral control. Both CD4⁺ and CD8⁺ T cells exhibited increased polyfunctionality after vaccination and was consistent across age groups. Single-cell multiomic analysis revealed comparable phenotypic composition and transcriptional profiles of global and preF-specific T cells between younger and older adults. Additionally, T-cell receptor repertoire analysis demonstrated clonal overlap in vaccine-induced CD4⁺ and CD8⁺ T cells across age groups, supporting a shared clonal architecture of the vaccine-elicited response. Our results provide strong evidence that mRNA-1345 elicits durable and polyfunctional T-cell immunity in adults across age groups, with the potential to confer long-term protection against RSV. Yanbo Sun, PhD, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds (Public Company) Emily Marcisak, MS, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds (Public Company) Daniel Makrinos, MS, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds (Public Company) Jenna Landy, BS, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds (Public Company) Shannon McGrath, MS, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds (Public Company) Hsiaohsuan Kuo, PhD, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds (Public Company) Maria Cavallaro, BS, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds (Public Company) Christopher Wu, MSc, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds (Public Company) Adam Essene, MS, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds (Public Company) Haining Lin, PhD, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds (Public Company) Wen-Han Yu, PhD, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds (Public Company) Anthony DiPiazza, PhD, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds (Public Company) Jaap Oostendorp, PhD, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds (Public Company) Robert Paris, MD, Moderna, Inc.: Employee|Moderna, Inc.: Stocks/Bonds (Public Company)

The local and systemic symptoms that follow vaccination, collectively referred to as reactogenicity, are common, yet the mechanisms underlying individual variability remain poorly understood. Through longitudinal immune profiling of vaccinated individuals and mechanistic studies in mice, we identified key immunological determinants of reactogenicity induced by mRNA vaccines. Systemic adverse events were associated with stronger interferon and pro-inflammatory responses after the second dose of a COVID-19 mRNA vaccine, which were also correlated with the magnitude of the antigen-specific adaptive responses. This heightened inflammation occurred within 24 hours of vaccination, originated primarily from the injection site, and was characterized by enhanced recruitment and activation of myeloid cells, particularly monocytes. Two mechanisms contributed to this response: early interferon production by muscle T cells generated after the first dose and Fcγ receptor-dependent chemokine induction by vaccine antigen-specific antibodies. Consistently, serum antibody levels before vaccination correlated positively with reactogenicity. In addition to this local amplification mechanism, variability in reactogenicity was influenced by the baseline immune state, given that individuals with a preexisting interferon-stimulated gene signature in monocytes, detectable at both transcriptomic and epigenetic levels, were more prone to systemic symptoms. Together, our findings revealed molecular and cellular mechanisms driving vaccine reactogenicity, providing a framework for the design of less reactogenic vaccines.

Influenza causes substantial morbidity and mortality worldwide. This randomized, open-label, phase 1 trial (ClinicalTrials.gov, NCT05397223, date of registration: May 31, 2022) compared the immunogenicity of an mRNA-based quadrivalent influenza hemagglutinin (HA) vaccine (mRNA-1010) with a licensed comparator (FLUAD) in adults aged 18-75 years. We evaluated humoral and cellular immune responses using hemagglutination inhibition assays, flow cytometry-based memory B cell (MBC) profiling, and intracellular cytokine staining for T-cell characterization. Both vaccines elicited durable hemagglutination inhibition titers and increased HA-specific MBC responses across four vaccine strains. Compared with FLUAD, mRNA-1010 induced higher frequencies of classical and activated MBCs specific to the H3 HA included in the vaccine, while inducing similar MBC responses to the other strains. mRNA-1010 and FLUAD generated strong HA-specific CD4+ T-cell responses; a trend toward higher CD8+ T-cell responses was observed in mRNA-1010 recipients compared with FLUAD recipients for two of the four strains. These findings support the potential of the mRNA platform for seasonal influenza vaccination.

Introduction:Clinical trials do not typically assess underlying molecular mechanisms of vaccine immunogenicity or reactogenicity. We evaluated the reactogenicity and immunogenicity of 4 mRNA vaccines and potential contributing mechanisms and identified shared and unique clinical and immunologic features. Methods:This ongoing, open-label, phase 1 trial randomized healthy adults (18-75 years) to receive a single dose of mRNA-1273.222 (bivalent COVID-19), mRNA-1345 (RSV), mRNA-1010 (influenza), and FLUAD (active influenza comparator) or 2 or 3 doses of mRNA-1647 (CMV). The primary objective was to assess the safety and reactogenicity of each study vaccine, with humoral immunogenicity (neutralizing antibody [nAb] responses) as the secondary objective. This interim analysis reports safety and reactogenicity in all study vaccines and humoral immunogenicity in single-dose vaccines (mRNA-1273.222, mRNA-1345, mRNA-1010, and FLUAD). Exploratory objectives included antigen-specific T-cell responses after single-dose mRNA-1345 or mRNA-1273.222, and soluble mediators of inflammation and innate immunity following vaccination in single-dose vaccine groups and two doses of mRNA-1647. Results:At the interim analysis data cutoff (February 1, 2023), 302 participants received 1 dose of the study vaccines. Reactogenicity exhibited a consistent trend across vaccine groups; most solicited local and systemic adverse reactions within 7 days were mild or moderate in severity. There were no deaths or serious, severe, or treatment-related adverse events leading to study discontinuation. At Day 29, nAb titers against vaccine-specific antigens increased 2- to 8-fold versus baseline for all single-dose vaccine groups. In an exploratory analysis, mRNA-1273.222 and mRNA-1345 induced antigen-specific Th1-biased CD4+ and CD8+ T-cell responses at Day 29. The cytokine response analysis showed increased levels of IFN-γ, IL-6, IL-2Ra, CXCL9, IP-10, MCP-2, and MIP-1β on Day 2 following vaccination, with generally greater increases observed with mRNA vaccines versus FLUAD. Regardless of age and across mRNA vaccine groups, peak serum levels of IL-1Ra and MCP-1/MCP-2 on Day 2 weakly correlated with systemic reactogenicity scores (correlation coefficient range: 0.15-0.27). Conclusions:The 4 mRNA vaccines had acceptable reactogenicity, demonstrated changes in serum biomarkers of innate immune activation, and were immunogenic. This suggests that the observed reactogenicity of mRNA vaccines may be related to shared features of the mRNA platform (LNP platform). Clinical trial registration:ClinicalTrials.gov, identifier NCT05397223.

Chronic hepatitis B remains a major global health challenge, affecting over 254 million individuals and causing over 1 million deaths annually. Despite current antiviral therapies effectively suppressing viral replication, functional cure rates are low due to HBV-induced immune dysfunction and exhaustion. Therefore, new therapeutic approaches to achieve immune control of HBV infection are needed. Following the systematic evaluation of multiple HBV mRNA antigen designs, we developed mRNA-1965, a trivalent therapeutic mRNA vaccine encoding nanoparticle-displayed PreS1 and PreS2 domains of HBsAg to bypass the immune interference caused by HBV subviral particles, along with mutant forms of HBV Core and Polymerase. In HBV naïve mice, mRNA-1965 immunization induced dose-dependent HBV-neutralizing antibodies and Th1-skewed CD4+ and IFNγ+ CD8+ T cell responses to all three encoded HBV antigens. In non-human primates, mRNA-1965 elicited broad antibody and T cell responses across multiple HBV genotypes. Furthermore, vaccination with mRNA-1965 achieved a strong neutralizing antibody response and complete clearance of serum and liver HBV biomarkers in in an AAV-HBV mouse model with ∼100 IU/mL baseline HBsAg. Notably, combining mRNA-1965 with immune stimulatory co-modalities targeting PD-L1 and OX40 further enhanced therapeutic efficacy in mice with ∼1000 IU/mL baseline HBsAg. Clearance of HBV in AAV-HBV mice was associated with T cell response to mRNA-encoded antigens and with activation and differentiation of Core-specific CD8+ T cells. These findings support the potential of mRNA-1965 to promote a functional cure for chronic hepatitis B by overcoming immune dysfunction and subsequently enabling robust, functional immunity.

ABSTRACT mRNA-1647 is an investigational mRNA-based vaccine against cytomegalovirus (CMV) that contains sequences encoding the CMV proteins glycoprotein B and pentamer. Humoral and cellular immune responses were evaluated in blood samples collected from healthy CMV-seropositive and CMV-seronegative adults who participated in a phase 1 trial of a three-dose series of mRNA-1647 (NCT03382405). Neutralizing antibody (nAb) titers against fibroblast and epithelial cell infection in sera from CMV-seronegative mRNA-1647 recipients were higher than those in sera from control CMV-seropositive samples and remained elevated up to 12 months after dose 3. nAb responses elicited by mRNA-1647 were comparable across 14 human CMV (HCMV) strains. Frequencies of antigen-specific memory B cells increased in CMV-seropositive and CMV-seronegative participants after each mRNA-1647 dose and remained elevated for up to 6 months after dose 3. mRNA-1647 elicited robust increases in frequencies and polyfunctionality of CD4 + T helper type 1 and effector CD8 + T cells in samples from CMV-seronegative and CMV-seropositive participants after stimulation with HCMV-specific peptides. The administration of three doses of mRNA-1647 to healthy adults elicited high nAb titers with wide-breadth, long-lasting memory B cells, and strong polyfunctional T-cell responses. These findings support further clinical development of the mRNA-1647 vaccine against CMV. IMPORTANCE Cytomegalovirus (CMV), a common virus that can infect people of all ages, may lead to serious health problems in unborn babies and those with a weakened immune system. Currently, there is no approved vaccine available to prevent CMV infection; however, the investigational messenger RNA (mRNA)–based CMV vaccine, mRNA-1647, is undergoing evaluation in clinical trials. The current analysis examined samples from a phase 1 trial of mRNA-1647 in healthy adults to better understand how the immune system reacts to vaccination. Three doses of mRNA-1647 produced a long-lasting immune response, thus supporting further investigation of the vaccine in the prevention of CMV infection. CLINICAL TRIALS Registered at ClinicalTrials.gov ( NCT03382405 ).

Increased use of antiviral monoclonal antibodies (mAbs) for treatment and prophylaxis necessitates better understanding of their impact on endogenous immunity to vaccines and viruses. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic presented an opportunity to study immunity in individuals who received antiviral mAbs and were subsequently immunized with vaccines encoding the mAb-targeted viral spike antigen. Here, we describe the impact of administration of an antibody combination, casirivimab plus imdevimab (CAS+IMD), on immune responses to subsequent SARS-CoV-2 vaccination in humans, nonhuman primates, and mice. The presence of CAS+IMD at the time of vaccination led to a specific diminishment of vaccine-elicited pseudovirus neutralizing antibody titers without overall dampening of spike protein-directed immune responses, including antibody, B cell, and T cell responses. The impact on pseudovirus neutralizing titers extended to other therapeutic anti-spike protein antibodies when used as either monotherapy or combination therapy. The specific reduction in pseudovirus neutralizing titers was the result of epitope masking, a phenomenon where specific epitopes are bound by high-affinity antibodies and blocked from B cell recognition. Encouragingly, this reduction in pseudovirus neutralizing titers was reversible with additional booster vaccination. Moreover, by assessing the antiviral immune response in SARS-CoV-2-infected individuals treated therapeutically with CAS+IMD, we demonstrated alteration of antiviral humoral immunity in those who had received mAb therapy, but only in those individuals who had yet to start mounting their natural immune response at the time of mAb treatment. Together, these data demonstrate that antiviral mAbs can alter endogenous humoral immunity during vaccination or infection.

For a vaccine to achieve durable immunity and optimal efficacy, many require a multi-dose primary vaccination schedule that acts to first “prime” naive immune systems and then “boost” initial immune responses by repeated immunizations (ie, prime-boost regimens). In the context of the global coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 2-dose primary vaccination regimens were often selected with short intervals between doses to provide rapid protection while still inducing robust immunity. However, emerging post-authorization evidence has suggested that longer intervals between doses 1 and 2 for SARS-CoV-2 vaccines may positively impact robustness and durability of immune responses. Here, the dosing interval for mRNA-1273, a messenger RNA based SARS-CoV-2 vaccine administered on a 2-dose primary schedule with 4 weeks between doses, was evaluated in mice by varying the dose interval between 1 and 8 weeks and examining immune responses through 24 weeks after dose 2. A dosing interval of 6 to 8 weeks generated the highest level of antigen-specific serum immunoglobulin G binding antibody titers. Differences in binding antibody titers between mRNA-1273 1 µg and 10 µg decreased over time for dosing intervals of ≥4 weeks, suggesting a potential dose-sparing effect. Longer intervals (≥4 weeks) also increased antibody-dependent cellular cytotoxicity activity and numbers of antibody-secreting cells (including long-lived plasma cells) after the second dose. An interval of 6 to 8 weeks elicited the strongest CD8+ T-cell responses, while an interval of 3 weeks elicited the strongest CD4+ T-cell response. Overall, these results suggest that in a non-pandemic setting, a longer interval (≥6 weeks) between the doses of the primary series for mRNA-1273 may induce more durable immune responses.

The emergence of highly transmissible SARS-CoV-2 variants of concern (VOC) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identify four receptor-binding domain targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 12 variants including the B.1.1.7 and B.1.351 VOCs. Two of them are ultrapotent, with sub-nanomolar neutralization titers (IC50 <0.0006 to 0.0102 μ g/mL; IC80 < 0.0006 to 0.0251 μ g/mL). We define the structural and functional determinants of binding for all four VOC-targeting antibodies, and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting potential means to mitigate resistance development. These results define the basis of therapeutic cocktails against VOCs and suggest that targeted boosting of existing immunity may increase vaccine breadth against VOCs.

Vaccine-associated enhanced respiratory disease (VAERD) was previously observed in some preclinical models of severe acute respiratory syndrome (SARS) and MERS coronavirus vaccines. We used the SARS coronavirus 2 (SARS-CoV-2) mouse-adapted, passage 10, lethal challenge virus (MA10) mouse model of acute lung injury to evaluate the immune response and potential for immunopathology in animals vaccinated with research-grade mRNA-1273. Whole-inactivated virus or heat-denatured spike protein subunit vaccines with alum designed to elicit low-potency antibodies and Th2-skewed CD4(+) T cells resulted in reduced viral titers and weight loss post challenge but more severe pathological changes in the lung compared to saline-immunized animals. In contrast, a protective dose of mRNA-1273 induced favorable humoral and cellular immune responses that protected from viral replication in the upper and lower respiratory tract upon challenge. A subprotective dose of mRNA-1273 reduced viral replication and limited histopathological manifestations compared to animals given saline. Overall, our findings demonstrate an immunological signature associated with antiviral protection without disease enhancement following vaccination with mRNA-1273.

SARS-CoV-2 first emerged in the human population in late 2019 in Wuhan, China, and in a matter of months, spread across the globe resulting in the Coronavirus Disease 19 (COVID-19) pandemic and substantial economic fallout. SARS-CoV-2 is transmitted between humans via respiratory particles, with infection presenting a spectrum of clinical manifestations ranging from asymptomatic to respiratory failure with multiorgan dysfunction and death in severe cases. Prior experiences with human pathogenic coronaviruses and respiratory virus diseases in general have revealed an important role for cellular immunity in limiting disease severity. Here, we review some of the key mechanisms underlying cell-mediated immunity to respiratory viruses and summarize our current understanding of the functional capacity and role of SARS-CoV-2-specific T cells following natural infection and vaccination.

Nipah virus (NiV) represents a significant pandemic threat with zoonotic transmission from bats-to-humans with almost annual regional outbreaks characterized by documented human-to-human transmission and high fatality rates. Currently, no vaccine against NiV has been approved. Structure-based design and protein engineering principles were applied to stabilize the fusion (F) protein in its prefusion trimeric conformation (pre-F) to improve expression and increase immunogenicity. We covalently linked the stabilized pre-F through trimerization domains at the C-terminus to three attachment protein (G) monomers, forming a chimeric design. These studies detailed here focus on mRNA delivery of NiV immunogens in mice, assessment of mRNA immunogen-specific design elements and their effects on humoral and cellular immunogenicity. The pre-F/G chimera elicited a strong neutralizing antibody response and a superior NiV-specific Tfh and other effector T cell response compared to G alone across both the mRNA and protein platforms. These findings enabled final candidate selection of pre-F/G Fd for clinical development.

The emergence of highly transmissible SARS-CoV-2 variants of concern (VOCs) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identified four receptor binding domain-targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 23 variants, including the B.1.1.7, B.1.351, P.1, B.1.429, B.1.526, and B.1.617 VOCs. Two antibodies are ultrapotent, with subnanomolar neutralization titers [half-maximal inhibitory concentration (IC50) 0.3 to 11.1 nanograms per milliliter; IC80 1.5 to 34.5 nanograms per milliliter). We define the structural and functional determinants of binding for all four VOC-targeting antibodies and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting their potential in mitigating resistance development.

A vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to control the coronavirus disease 2019 (COVID-19) global pandemic. Structural studies have led to the development of mutations that stabilize Betacoronavirus spike proteins in the prefusion state, improving their expression and increasing immunogenicity(1). This principle has been applied to design mRNA-1273, an mRNA vaccine that encodes a SARS-CoV-2 spike protein that is stabilized in the prefusion conformation. Here we show that mRNA-1273 induces potent neutralizing antibody responses to both wild-type (D614) and D614G mutant(2) SARS-CoV-2 as well as CD8(+) T cell responses, and protects against SARS-CoV-2 infection in the lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a phase III trial to evaluate its efficacy.

Biotin-labeled molecular probes, comprising specific regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. Here, we design constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions include full-length spike ectodomain as well as various subregions, and we also design mutants that eliminate recognition of the angiotensin-converting enzyme 2 (ACE2) receptor. Yields of biotin-labeled probes from transient transfection range from ∼0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes are characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe is determined by cryoelectron microscopy. We also characterize antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike ectodomain probes.

Technologies that define the atomic-level structure of neutralization-sensitive epitopes on viral surface proteins are transforming vaccinology and guiding new vaccine development approaches. Previously, iterative rounds of protein engineering were performed to preserve the prefusion conformation of the respiratory syncytial virus (RSV) fusion (F) glycoprotein, resulting in a stabilized subunit vaccine candidate (DS-Cav1), which showed promising results in mice and macaques. Here, phase I human immunogenicity data reveal a more than 10-fold boost in neutralizing activity in serum from antibodies targeting prefusion-specific surfaces of RSV F. These findings represent a clinical proof of concept for structure-based vaccine design, suggest that development of a successful RSV vaccine will be feasible, and portend an era of precision vaccinology.

This 20-color flow cytometry panel was designed to resolve the cellular heterogeneity of antigen-presenting cells and was optimized for lymph node tissue. Reagents were carefully selected and optimized for identification of B cells (B220), neutrophils (Ly6G), monocytes and macrophages (Ly6C, CD169, F4/80), and dendritic cells (XCR1, CD172a, CD11c, I-A/I-E, CD24, CD64, pDCA-1, CD103, CD11b). Inclusion of additional functional markers involved in cell migration (CCR7), co-stimulation (CD80), and adhesion (ICAM-1) enabled further phenotypic characterization. Finally, this panel has been tested and is compatible with fluorescently labeled antigens such as Alexa Fluor 488 (Ax488) for the study of antigen-bearing cells in vivo. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.

Avian influenza vaccines exhibit poor immunogenicity in humans. We hypothesized that one factor underlying weak B cell responses was sequence divergence between avian and seasonal influenza hemagglutinin proteins, thus limiting the availability of adequate CD4 T cell help. To test this, a novel chimeric hemagglutinin protein (cH7/3) was derived, comprised of the stem domain from seasonal H3 hemagglutinin and the head domain from avian H7. Immunological memory to seasonal influenza was established in mice, through strategies that included seasonal inactivated vaccines, Flumist, and synthetic peptides derived from the H3 stalk domain. After establishment of memory, mice were vaccinated with H7 or cH7/3 protein. The cH7/3 Ag was able to recall H3-specific CD4 T cells, and this potentiated CD4 T cell response was associated with enhanced early germinal center response and rapid elicitation of Abs to H7, including Abs specific for the H7 head domain. These results suggest that in pandemic situations, inclusion of CD4 T cell epitopes from seasonal viruses have the potential to overcome the poor immunogenicity of avian vaccines by helping B cells and conferring greater subtype-specific Ab response to viral HA.

CD4 T cells convey a number of discrete functions to protective immunity to influenza, a complexity that distinguishes this arm of adaptive immunity from B cells and CD8 T cells. Although the most well recognized function of CD4 T cells is provision of help for antibody production, CD4 T cells are important in many aspects of protective immunity. Our studies have revealed that viral antigen specificity is a key determinant of CD4 T cell function, as illustrated both by mouse models of infection and human vaccine responses, a factor whose importance is due at least in part to events in viral antigen handling. We discuss research that has provided insight into the diverse viral epitope specificity of CD4 T cells elicited after infection, how this primary response is modified as CD4 T cells home to the lung, establish memory, and after challenge with a secondary and distinct influenza virus strain. Our studies in human subjects point out the challenges facing vaccine efforts to facilitate responses to novel and avian strains of influenza, as well as strategies that enhance the ability of CD4 T cells to promote protective antibody responses to both seasonal and potentially pandemic strains of influenza.

Ferrets are an ideal animal model in which to study the transmission of respiratory viruses as well as disease progression and vaccine efficacy because of their close anatomical and physiological resemblances to humans. However, a paucity of reagents and standardized procedures has hampered research progress, especially for studying cell-mediated immunity. The approaches described here-leukocyte isolation from whole blood and secondary lymphoid tissues-are generalizable, highly reproducible, and deliver single cell suspensions with excellent cell viability. Importantly, we have now developed assays to quantify key cellular components and antigen-specific T cell responses at the single cell level from multiple tissue compartments following influenza infection in ferrets. Collectively, these methods were instrumental in flow cytometry studies that revealed alterations in immune cell composition and distribution across lymphoid tissues following viral infection. Furthermore, sorting of T cell populations and peptide restimulation ex vivo in cytokine ELISpot assays has provided novel insight into the influenza-specific CD4 and CD8 T cell repertoire. The detailed procedures for these techniques are described in this chapter and can likely be adapted for the analyses of responses to many respiratory pathogens.