Jennifer L. Nayak

CD4+ T-cell expansion predicts neutralizing antibody responses to monovalent, inactivated 2009 pandemic influenza A(H1N1) virus subtype H1N1 vaccine.

Abstract Background. The ability of influenza vaccines to elicit CD4+ T cells and the relationship between induction of CD4+ T cells and vaccine-induced neutralizing antibody responses has been controversial. The emergence of swine-origin 2009 pandemic influenza A virus subtype H1N1 (A[H1N1]pdm09) provided a unique opportunity to examine responses to an influenza vaccine composed of both novel and previously encountered antigens and to probe the relationship between B-cell and T-cell responses to vaccination. Methods. We tracked CD4+ T-cell and antibody responses of human subjects vaccinated with monovalent subunit A(H1N1)pdm09 vaccine. The specificity and magnitude of the CD4+ T-cell response was evaluated using cytokine enzyme-linked immunosorbent spot assays in conjugation with peptide pools representing distinct influenza virus proteins. Results. Our studies revealed that vaccination induced readily detectable CD4+ T cells specific for conserved portions of hemagglutinin (HA) and the internal viral proteins. Interestingly, expansion of HA-specific CD4+ T cells was most tightly correlated with the antibody response. Conclusions. These results indicate that CD4+ T-cell expansion may be a limiting factor in development of neutralizing antibody responses to pandemic influenza vaccines and suggest that approaches to facilitate CD4+ T-cell recruitment may increase the neutralizing antibody produced in response to vaccines against novel influenza strains.

Analyses of the Specificity of CD4 T Cells During the Primary Immune Response to Influenza Virus Reveals Dramatic MHC-Linked Asymmetries in Reactivity to Individual Viral Proteins

Influenza is a contagious, acute respiratory disease that is a major cause of morbidity and mortality throughout the world. CD4 T cells play an important role in the immune response to this pathogen through the secretion of antiviral cytokines, and by providing help to CD8 T cells and B cells to promote the development of immunological memory and neutralizing antibody responses. Despite these well-defined roles in the anti-influenza response, our understanding of CD4 T-cell diversity and specificity remains limited. In the study reported here, overlapping peptides representing 5 different influenza viral proteins were used in EliSpot assays to enumerate and identify the specificity of anti-influenza CD4 T cells directly ex vivo following infection of mice with influenza virus, using two strains that express unrelated MHC class II molecules. These experiments evaluated whether the reactivity of CD4 T cells generally tracked with particular influenza proteins, or whether MHC preferences were the predominant factor dictating anti-CD4 T-cell specificity in the primary immune response. We made the unexpected discovery that the distribution of CD4 T-cell specificities for different influenza proteins varied significantly depending on the single class II molecule expressed in vivo. In SJL mice, the majority of epitopes were specific for the HA protein, while the NP protein dominated the response in C57BL/10 mice. Given the diversity of human MHC class II molecules, these findings have important implications for the ability to rationally design a vaccine that will generate a specific CD4 T-cell immune response that is effective across diverse human populations.

The utility and limitations of current Web-available algorithms to predict peptides recognized by CD4 T cells in response to pathogen infection.

The ability to track CD4 T cells elicited in response to pathogen infection or vaccination is critical because of the role these cells play in protective immunity. Coupled with advances in genome sequencing of pathogenic organisms, there is considerable appeal for implementation of computer-based algorithms to predict peptides that bind to the class II molecules, forming the complex recognized by CD4 T cells. Despite recent progress in this area, there is a paucity of data regarding the success of these algorithms in identifying actual pathogen-derived epitopes. In this study, we sought to rigorously evaluate the performance of multiple Web-available algorithms by comparing their predictions with our results—obtained by purely empirical methods for epitope discovery in influenza that used overlapping peptides and cytokine ELISPOTs—for three independent class II molecules. We analyzed the data in different ways, trying to anticipate how an investigator might use these computational tools for epitope discovery. We come to the conclusion that currently available algorithms can indeed facilitate epitope discovery, but all shared a high degree of false-positive and false-negative predictions. Therefore, efficiencies were low. We also found dramatic disparities among algorithms and between predicted IC50 values and true dissociation rates of peptide–MHC class II complexes. We suggest that improved success of predictive algorithms will depend less on changes in computational methods or increased data sets and more on changes in parameters used to “train” the algorithms that factor in elements of T cell repertoire and peptide acquisition by class II molecules.

Effect of Influenza A(H5N1) Vaccine Prepandemic Priming on CD4+ T-Cell Responses

Abstract Introduction. Previous priming with avian influenza vaccines results in more rapid and more robust neutralizing antibody responses upon revaccination, but the role CD4+ T cells play in this process is not currently known. Methods. Human subjects previously enrolled in trials of inactivated influenza A(H5N1) vaccines and naive subjects were immunized with an inactivated subunit influenza A/Indonesia/5/05(H5N1) vaccine. Neutralizing antibody responses were measured by a microneutralization assay, and hemagglutinin (HA)-specific and nucleoprotein (NP)-specific CD4+ T-cell responses were quantified using interferon γ enzyme-linked immunosorbent spot assays. Results. While vaccination induced barely detectable CD4+ T-cell responses specific for HA in the previously unprimed group, primed subjects had readily detectable HA-specific memory CD4+ T cells at baseline and mounted a more robust response to HA-specific epitopes after vaccination. There were no differences between groups when conserved NP-specific CD4+ T-cell responses were examined. Interestingly, neutralizing antibody responses following revaccination were significantly higher in individuals who mounted a CD4+ T-cell response to the H5 HA protein, a correlation not observed for NP-specific responses. Conclusions. These findings suggest that prepandemic vaccination results in an enriched population of HA-specific CD4+ T cells that are recruited on rechallenge with a drifted vaccine variant and contribute to more robust and more rapid neutralizing antibody responses.

Seasonal Influenza Can Poise Hosts for CD4 T-Cell Immunity to H7N9 Avian Influenza

The emergence of avian H7N9 viruses has raised concerns about its pandemic potential and prompted vaccine trials. At present, it is unknown whether there will be sufficient cross-reactive hemagglutinin (HA)-specific CD4 T-cell memory with seasonal influenza to facilitate antibody production to H7 HA. There has also been speculation that H7N9 will have few CD4 T-cell epitopes. In this study, we quantified the potential of seasonal influenza to provide memory CD4 T cells that can cross-reactively recognize H7 HA-derived peptides. These studies have revealed that many humans have substantial H7-reactive CD4 T cells, whereas up to 40% are lacking such reactivity. Correlation studies indicate that CD4 T cells reactive with H7 HA are drawn from reactivity generated from seasonal strains. Overall, our findings suggest that previous exposure of humans to seasonal influenza can poise them to respond to avian H7N9, but this is likely to be uneven across populations.

The Role of CD4 T Cell Memory in Generating Protective Immunity to Novel and Potentially Pandemic Strains of Influenza.

Recent events have made it clear that potentially pandemic strains of influenza regularly pose a threat to human populations. Therefore, it is essential that we develop better strategies to enhance vaccine design and evaluation to predict those that will be poor responders to vaccination and to identify those that are at particular risk of disease-associated complications following infection. Animal models have revealed the discrete functions that CD4 T cells play in developing immune response and to influenza immunity. However, humans have a complex immunological history with influenza through periodic infection and vaccination with seasonal variants, leading to the establishment of heterogeneous memory populations of CD4 T cells that participate in subsequent responses. The continual evolution of the influenza-specific CD4 T cell repertoire involves both specificity and function and overlays other restrictions on CD4 T cell activity derived from viral antigen handling and MHC class II:peptide epitope display. Together, these complexities in the influenza-specific CD4 T cell repertoire constitute a formidable obstacle to predicting protective immune response to potentially pandemic strains of influenza and in devising optimal vaccine strategies to potentiate these responses. We suggest that more precise efforts to identify and enumerate both the positive and negative contributors within the CD4 T cell compartment will aid significantly in the achievement of these goals.

Cutting Edge: Heterosubtypic Influenza Infection Antagonizes Elicitation of Immunological Reactivity to Hemagglutinin

Influenza-specific immunity in humans is unique because there are repeated exposures to viral strains containing genetically conserved epitopes recruiting memory CD4 T cells and novel epitopes stimulating naive CD4 T cells, possibly resulting in competition between memory and naive lymphocytes. In this study, we evaluated the effect of this competition on CD4 T cell and B cell response specificity using a murine model of sequential influenza infection. We found striking and selective decreases in CD4 T cell reactivity to nonconserved hemagglutinin (HA) epitopes following secondary influenza infection. Surprisingly, this shift in CD4 T cell specificity was associated with dramatic decreases in HA-specific Ab. These results suggest that repeated exposure to influenza viruses and vaccines containing conserved internal proteins may have unintended and negative consequences on the ability to induce HA-specific Ab to novel pandemic strains of influenza. These finding could have important implications on pandemic influenza preparedness strategies.

Selective pre-priming of HA-specific CD4 T cells restores immunological reactivity to HA on heterosubtypic influenza infection

A hallmark of the immune response to influenza is repeated encounters with proteins containing both genetically conserved and variable components. Therefore, the B and T cell repertoire is continually being remodeled, with competition between memory and naïve lymphocytes. Our previous work using a mouse model of secondary heterosubtypic influenza infection has shown that this competition results in a focusing of CD4 T cell response specificity towards internal virion proteins with a selective decrease in CD4 T cell reactivity to the novel HA epitopes. Strikingly, this shift in CD4 T cell specificity was associated with a diminished anti-HA antibody response. Here, we sought to determine whether the loss in HA-specific reactivity that occurs as a consequence of immunological memory could be reversed by selectively priming HA-specific CD4 T cells prior to secondary infection. Using a peptide-based priming strategy, we found that selective expansion of the anti-HA CD4 T cell memory repertoire enhanced HA-specific antibody production upon heterosubtypic infection. These results suggest that the potentially deleterious consequences of repeated exposure to conserved influenza internal virion proteins could be reversed by vaccination strategies that selectively arm the HA-specific CD4 T cell compartment. This could be a potentially useful pre-pandemic vaccination strategy to promote accelerated neutralizing antibody production on challenge with a pandemic influenza strain that contains few conserved HA epitopes.

Immunization with Pneumocystis Cross-Reactive Antigen 1 (Pca1) Protects Mice against Pneumocystis Pneumonia and Generates Antibody to Pneumocystis jirovecii

ABSTRACT Pneumocystis pneumonia (PcP) is a life-threatening infection that affects immunocompromised individuals. Nearly half of all PcP cases occur in those prescribed effective chemoprophylaxis, suggesting that additional preventive methods are needed. To this end, we have identified a unique mouse Pneumocystis surface protein, designated Pneumocystis cross-reactive antigen 1 (Pca1), as a potential vaccine candidate. Mice were immunized with a recombinant fusion protein containing Pca1. Subsequently, CD4+ T cells were depleted, and the mice were exposed to Pneumocystis murina. Pca1 immunization completely protected nearly all mice, similar to immunization with whole Pneumocystis organisms. In contrast, all immunized negative-control mice developed PcP. Unexpectedly, Pca1 immunization generated cross-reactive antibody that recognized Pneumocystis jirovecii and Pneumocystis carinii. Potential orthologs of Pca1 have been identified in P. jirovecii. Such cross-reactivity is rare, and our findings suggest that Pca1 is a conserved antigen and potential vaccine target. The evaluation of Pca1-elicited antibodies in the prevention of PcP in humans deserves further investigation.

Distinct and complementary roles of CD4 T cells in protective immunity to influenza virus

CD4 T cells play a multiplicity of roles in protective immunity to influenza. Included in these functions are help for high affinity antibody production, enhancement of CD8 T cell expansion, function and memory, acceleration of the early innate response to infection and direct cytotoxicity. The influenza-specific CD4 T cell repertoire in humans established through exposures to infection and vaccination has been found to be highly variable in abundance, specificity and functionality. Deficits in particular subsets of CD4 T cells recruited into the response result in diminished antibody responses and protection from infection. Therefore, improved strategies for vaccination should include better methods to identify deficiencies in the circulating CD4 T cell repertoire, and vaccine constructs that increase the representation of CD4 T cells of the correct specificity and functionality.