Induction of heterosubtypic immunity to influenza virus by intranasal immunization

Recovery from live influenza virus infection is known to induce heterosubtypic immunity. In contrast, immunity induced by inactivated vaccines is predominantly subtype specific. In this study, we investigated the heterosubtypic protective immunity induced by inactivated influenza virus. Intranasal immunization of mice with inactivated influenza virus A/PR8 (H1N1) provided complete protection against the homologous virus and a drift virus within the same subtype, A/WSN (H1N1), but not against the heterosubtypic virus A/Philippines (H3N2). However, coadministration of inactivated virus with cholera toxin as an adjuvant conferred complete heterosubtypic protection, without observed illness, even under conditions of CD4⁺ or CD8⁺ T-cell depletion. Analysis of immune correlates prior to challenge and postchallenge indicated that humoral immune responses with cross-neutralizing activity in lungs and in sera play a major role in conferring protective immunity against heterosubtypic challenge. This study has significant implications for developing broadly cross-reactive vaccines against newly emerging pathogenic influenza viruses.     

Clearance of an Influenza A Virus by CD4+ T Cells Is Inefficient in the Absence of B Cells

The primary CD8⁺ T-cell response protected most B-cell-deficient μMT mice against intranasal infection with the HKx31 influenza A virus. Prior exposure did not prevent reinfection upon homologous challenge, and the recall CD8⁺ T-cell response cleared the virus from the lung within 7 days. Depleting the CD8⁺ T cells substantially reduced the capacity of these primed mice to deal with the infection, in spite of evidence for established CD4⁺ T-cell memory. Thus, the control of this relatively mild influenza virus by both primary and secondary CD4⁺ T-cell responses is relatively inefficient in the absence of B cells and CD8⁺ T cells.     

Protection against a Lethal Avian Influenza A Virus in a Mammalian System

The question of how best to protect the human population against a potential influenza pandemic has been raised by the recent outbreak caused by an avian H5N1 virus in Hong Kong. The likely strategy would be to vaccinate with a less virulent, laboratory-adapted H5N1 strain isolated previously from birds. Little attention has been given, however, to dissecting the consequences of sequential exposure to serologically related influenza A viruses using contemporary immunology techniques. Such experiments with the H5N1 viruses are limited by the potential risk to humans. An extremely virulent H3N8 avian influenza A virus has been used to infect both immunoglobulin-expressing (Ig^+/+) and Ig^−/− mice primed previously with a laboratory-adapted H3N2 virus. The cross-reactive antibody response was very protective, while the recall of CD8⁺ T-cell memory in the Ig^−/− mice provided some small measure of resistance to a low-dose H3N8 challenge. The H3N8 virus also replicated in the respiratory tracts of the H3N2-primed Ig^+/+ mice, generating secondary CD8⁺ and CD4⁺ T-cell responses that may contribute to recovery. The results indicate that the various components of immune memory operate together to provide optimal protection, and they support the idea that related viruses of nonhuman origin can be used as vaccines.     

Reduced protection from simian immunodeficiency virus SIVmac251 infection afforded by memory CD8+ T cells induced by vaccination during CD4+ T-cell deficiency

Adaptive CD4⁺ and CD8⁺ T-cell responses have been associated with control of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) replication. Here, we have designed a study with Indian rhesus macaques to more directly assess the role of CD8 SIV-specific responses in control of viral replication. Macaques were immunized with a DNA prime-modified vaccinia virus Ankara (MVA)-SIV boost regimen under normal conditions or under conditions of antibody-induced CD4⁺ T-cell deficiency. Depletion of CD4⁺ cells was performed in the immunized macaques at the peak of SIV-specific CD4⁺ T-cell responses following the DNA prime dose. A group of naïve macaques was also treated with the anti-CD4 depleting antibody as a control, and an additional group of macaques immunized under normal conditions was depleted of CD8⁺ T cells prior to challenge exposure to SIV_mac251. Analysis of the quality and quantity of vaccine-induced CD8⁺ T cells demonstrated that SIV-specific CD8⁺ T cells generated under conditions of CD4⁺ T-cell deficiency expressed low levels of Bcl-2 and interleukin-2 (IL-2), and plasma virus levels increased over time. Depletion of CD8⁺ T cells prior to challenge exposure abrogated vaccine-induced protection as previously shown. These data support the notion that adaptive CD4⁺ T cells are critical for the generation of effective CD8⁺ T-cell responses to SIV that, in turn, contribute to protection from AIDS. Importantly, they also suggest that long-term protection from disease will be afforded only by T-cell vaccines for HIV that provide a balanced induction of CD4⁺ and CD8⁺ T-cell responses and protect against early depletion of CD4⁺ T cells postinfection.     

A mutation in the HLA-B*2705-restricted NP383-391 epitope affects the human influenza A virus-specific cytotoxic T-lymphocyte response in vitro

Viruses can exploit a variety of strategies to evade immune surveillance by cytotoxic T lymphocytes (CTL), including the acquisition of mutations in or adjacent to CTL epitopes. Recently, an amino acid substitution (R384G) in an HLA-B^*2705-restricted CTL epitope in the influenza A virus nucleoprotein (nucleoprotein containing residues 383 to 391 [NP_383-391]; SRYWAIRTR, where R is the residue that was mutated) was associated with escape from CTL-mediated immunity. The effect of this mutation on the in vitro influenza A virus-specific CTL response was studied. To this end, two influenza A viruses, one with and one without the NP_383-391 epitope, were constructed by reverse genetics and designated influenza viruses A/NL/94-384R and A/NL/94-384G, respectively. The absence of the HLA-B^*2705-restricted CTL epitope in influenza virus A/NL/94-384G was confirmed by using ⁵¹Cr release assays with a T-cell clone specific for the NP_383-391 epitope. In addition, peripheral blood mononuclear cells (PBMC) stimulated with influenza virus A/NL/94-384G failed to recognize HLA-B^*2705-positive target cells pulsed with the original NP_383-391 peptide. The proportion of virus-specific CD8⁺ gamma interferon (IFN-γ)-positive T cells in in vitro-stimulated PBMC was determined by intracellular IFN-γ staining after restimulation with virus-infected autologous B-lymphoblastoid cell lines and C1R cell lines expressing only HLA-B^*2705. The proportion of virus-specific CD8⁺ T cells was lower in PBMC stimulated in vitro with influenza virus A/NL/94-384G obtained from several HLA-B^*2705-positive donors than in PBMC stimulated with influenza virus A/NL/94-384R. This finding indicated that amino acid variations in CTL epitopes can affect the virus-specific CTL response and that the NP_383-391 epitope is the most important HLA-B^*2705-restricted epitope in the nucleoprotein of influenza A viruses.     

Identification of CD8+ T Cell Epitopes in the West Nile Virus Polyprotein by Reverse-Immunology Using NetCTL

Background

West Nile virus (WNV) is a growing threat to public health and a greater understanding of the immune response raised against WNV is important for the development of prophylactic and therapeutic strategies.

Methodology/Principal Findings

In a reverse-immunology approach, we used bioinformatics methods to predict WNV-specific CD8⁺ T cell epitopes and selected a set of peptides that constitutes maximum coverage of 20 fully-sequenced WNV strains. We then tested these putative epitopes for cellular reactivity in a cohort of WNV-infected patients. We identified 26 new CD8⁺ T cell epitopes, which we propose are restricted by 11 different HLA class I alleles. Aiming for optimal coverage of human populations, we suggest that 11 of these new WNV epitopes would be sufficient to cover from 48% to 93% of ethnic populations in various areas of the World.

Conclusions/Significance

The 26 identified CD8⁺ T cell epitopes contribute to our knowledge of the immune response against WNV infection and greatly extend the list of known WNV CD8⁺ T cell epitopes. A polytope incorporating these and other epitopes could possibly serve as the basis for a WNV vaccine.     

Lymphocytic choriomeningitis virus infection yields overlapping CD4+ and CD8+ T-cell responses

Activation of CD4⁺ T cells helps establish and sustain other immune responses. We have previously shown that responses against a broad set of nine CD4⁺ T-cell epitopes were present in the setting of lymphocytic choriomeningitis virus (LCMV) Armstrong infection in the context of H-2^d. This is quite disparate to the H-2^b setting, where only two epitopes have been identified. We were interested in determining whether a broad set of responses was unique to H-2^d or whether additional CD4⁺ T-cell epitopes could be identified in the setting of the H-2^b background. To pursue this question, we infected C57BL/6 mice with LCMV Armstrong and determined the repertoire of CD4⁺ T-cell responses using overlapping 15-mer peptides corresponding to the LCMV Armstrong sequence. We confirmed positive responses by intracellular cytokine staining and major histocompatibility complex (MHC)-peptide binding assays. A broad repertoire of responses was identified, consisting of six epitopes. These epitopes originate from the nucleoprotein (NP) and glycoprotein (GP). Out of the six newly identified CD4⁺ epitopes, four of them also stimulate CD8⁺ T cells in a statistically significant manner. Furthermore, we assessed these CD4⁺ T-cell responses during the memory phase of LCMV Armstrong infection and after infection with a chronic strain of LCMV and determined that a subset of the responses could be detected under these different conditions. This is the first example of a broad repertoire of shared epitopes between CD4⁺ and CD8⁺ T cells in the context of viral infection. These findings demonstrate that immunodominance is a complex phenomenon in the context of helper responses.     

Targeting a Polyepitope Protein Incorporating Multiple Class II-Restricted Viral Epitopes to the Secretory/Endocytic Pathway Facilitates Immune Recognition by CD4+ Cytotoxic T Lymphocytes: a Novel Approach to Vaccine Design

The role of CD4⁺ and CD8⁺ cells in the generation of an effective immune response against viral infections is well established. Moreover, there is an increasing realization that subunit vaccines which include both CD4⁺- and CD8⁺-T-cell epitopes are highly effective in controlling viral infections, as opposed to those which are designed to activate a CD8⁺- or CD4⁺-T-cell response alone. One of the major limitations of epitope-based vaccines designed to stimulate virus-specific CD4⁺ T cells is that endogenously expressed class II-restricted minimal cytotoxic-T-lymphocyte (CTL) epitopes are poorly recognized by CD4⁺ CTLs. In the present study we attempted to enhance the efficiency of class II-restricted endogenous presentation of minimal class II-restricted CTL epitopes by specifically targeting a polyepitope protein to class II processing compartments through the endosomal and/or lysosomal pathway. A significantly enhanced stimulation of virus-specific CD4⁺-T-cell clones by antigen-presenting cells (APC) expressing the recombinant polyepitope protein targeted to the endocytic/secretory pathway was readily demonstrated in cytotoxicity assays. In addition, in vitro activation of Epstein-Barr virus- and influenza virus-specific CD4⁺ memory CTLs by the recombinant constructs encoding the polyepitope protein, specifically targeted to the lysosomal compartment, was also demonstrated. The enhanced stimulatory capacity of APC expressing a lysosome-targeted polyepitope protein has important implications for vaccine design.There is now increasing evidence to suggest that both CD4⁺ and CD8⁺ T cells are critical for the generation of an effective immune response against intracellular pathogens. Although both CD4⁺ and CD8⁺ T cells recognize nonnative forms of the antigen in association with major histocompatibility complex (MHC) molecules, the presentation of antigen to these two types of T lymphocytes occurs through distinct pathways (24). In fact, the disparity in antigen presentation to these T cells is not due to processing differences but rather reflects the differences in the capacities of class I and class II molecules to bind antigenic determinants in an intracellular compartment. Indeed, earlier studies have shown that for processing and interaction with MHC class II molecules, antigen expressed de novo needs to be targeted to an endosomal or lysosomal compartment (5). There are two major pathways by which antigens are targeted to these compartments. The traditional pathway involves the phagocytosis or endocytosis of exogenous antigens, followed by degradation by acid proteases in the endosomal or lysosomal compartments (3, 8, 26, 41). On the other hand, class II-restricted presentation of endogenously synthesized proteins mainly involves membrane antigens which are thought to enter the endosomal or lysosomal pathway by internalization from the cell surface (11). Although, in certain experimental systems, cytoplasmic and nuclear proteins may also enter this endogenous pathway, generally these proteins are targets for the class I processing pathway (9, 14, 20, 27).One of the major limitations of the epitope-based vaccines designed to stimulate virus-specific CD4⁺ T cells is that endogenously expressed class II-restricted minimal cytotoxic T-lymphocyte (CTL) epitopes are poorly recognized by CD4⁺ CTLs (2, 35, 38). Based on these observations, we reasoned that a molecular approach that directly routes these epitopes into the MHC class II pathway, such as the endocytic or lysosomal compartments, might facilitate endogenous presentation to CD4⁺ T cells. The lysosome-associated membrane protein (LAMP-1) and the invariant chain (Ii) are transmembrane proteins which are localized predominantly in the lysosomes and endosomes, respectively. The cytoplasmic domains of these proteins contain specific targeting signals that mediate their translocation to the specific compartments. We therefore designed a chimeric polyepitope construct capable of encoding multiple class II-restricted CTL epitopes from Epstein-Barr virus (EBV) and influenza virus linked to the cytoplasmic and/or transmembrane domains of LAMP-1 and the Ii protein, with the aim of targeting the epitopes to the endosomal and lysosomal compartments. The data presented in this study clearly demonstrate that if the endogenously synthesized polyepitope protein is targeted to the endocytic/secretory pathway, processing and presentation of all the epitopes are dramatically enhanced. More importantly, minimal epitope sequences, without any natural flanking sequences, were adequate for efficient stimulation of the virus-specific memory CTL response, a result that has important implications for epitope-based vaccine design.     

Protective Efficacy of Cross-Reactive CD8+ T Cells Recognising Mutant Viral Epitopes Depends on Peptide-MHC-I Structural Interactions and T Cell Activation Threshold

Emergence of a new influenza strain leads to a rapid global spread of the virus due to minimal antibody immunity. Pre-existing CD8⁺ T-cell immunity directed towards conserved internal viral regions can greatly ameliorate the disease. However, mutational escape within the T cell epitopes is a substantial issue for virus control and vaccine design. Although mutations can result in a loss of T cell recognition, some variants generate cross-reactive T cell responses. In this study, we used reverse genetics to modify the influenza NP_336–374 peptide at a partially-solvent exposed residue (N->A, NPN3A mutation) to assess the availability, effectiveness and mechanism underlying influenza-specific cross-reactive T cell responses. The engineered virus induced a diminished CD8⁺ T cell response and selected a narrowed T cell receptor (TCR) repertoire within two Vβ regions (Vβ8.3 and Vβ9). This can be partially explained by the H-2D^bNPN3A structure that showed a loss of several contacts between the NPN3A peptide and H-2D^b, including a contact with His155, a position known to play an important role in mediating TCR-pMHC-I interactions. Despite these differences, common cross-reactive TCRs were detected in both the naïve and immune NPN3A-specific TCR repertoires. However, while the NPN3A epitope primes memory T-cells that give an equivalent recall response to the mutant or wild-type (wt) virus, both are markedly lower than wt->wt challenge. Such decreased CD8⁺ responses elicited after heterologous challenge resulted in delayed viral clearance from the infected lung. Furthermore, mice first exposed to the wt virus give a poor, low avidity response following secondary infection with the mutant. Thus, the protective efficacy of cross-reactive CD8⁺ T cells recognising mutant viral epitopes depend on peptide-MHC-I structural interactions and functional avidity. Our study does not support vaccine strategies that include immunization against commonly selected cross-reactive variants with mutations at partially-solvent exposed residues that have characteristics comparable to NPN3A.     

Human Cytotoxic T-Lymphocyte Repertoire to Influenza A Viruses

The murine CD8⁺ cytotoxic-T-lymphocyte (CTL) repertoire appears to be quite limited in response to influenza A viruses. The CTL responses to influenza A virus in humans were examined to determine if the CTL repertoire is also very limited. Bulk cultures revealed that a number of virus proteins were recognized in CTL assays. CTL lines were isolated from three donors for detailed study and found to be specific for epitopes on numerous influenza A viral proteins. Eight distinct CD8⁺ CTL lines were isolated from donor 1. The proteins recognized by these cell lines included the nucleoprotein (NP), matrix protein (M1), nonstructural protein 1 (NS1), polymerases (PB1 and PB2), and hemagglutinin (HA). Two CD4⁺ cell lines, one specific for neuraminidase (NA) and the other specific for M1, were also characterized. These CTL results were confirmed by precursor frequency analysis of peptide-specific gamma interferon-producing cells detected by ELISPOT. The epitopes recognized by 6 of these 10 cell lines have not been previously described; 8 of the 10 cell lines were cross-reactive to subtype H1N1, H2N2, and H3N2 viruses, 1 cell line was cross-reactive to subtypes H1N1 and H2N2, and 1 cell line was subtype H1N1 specific. A broad CTL repertoire was detected in the two other donors, and cell lines specific for the NP, NA, HA, M1, NS1, and M2 viral proteins were isolated. These findings indicate that the human memory CTL response to influenza A virus is broadly directed to epitopes on a wide variety of proteins, unlike the limited response observed following infection of mice.     

Protective Role of Gamma Interferon during the Recall Response to Influenza Virus

During secondary immune responses to influenza virus, virus-specific T memory cells are a major source of gamma interferon (IFN-γ). We assessed the contribution of IFN-γ to heterologous protection against the A/WSN/33 (H1N1) virus of wild-type and IFN-γ−/− mice previously immunized with the A/HK/68 (H3N2) virus. The IFN-γ−/− mice displayed significantly reduced survival rates subsequent to a challenge with various doses of the A/WSN/33 virus. This was associated with an impaired ability of the IFN-γ−/− mice to completely clear the pulmonary virus by day 7 after the challenge, although significant reduction of the virus titers was noted. However, the IFN-γ−/− mice developed type A influenza virus cross-reactive cytotoxic T lymphocytes (CTLs) similar to the wild-type mice, as demonstrated by both cytotoxicity and a limiting-dilution assay for the estimation of CTL precursor frequency. The pulmonary recruitment of T cells in IFN-γ−/− mice was not dramatically affected, and the percentage of CD4⁺ and CD8⁺ T cells was similar to that of wild-type mice. The T cells from IFN-γ−/− mice did not display a significant switch toward a Th2 profile. Furthermore, the IFN-γ−/− mice retained the ability to mount significant titers of WSN and HK virus-specific hemagglutination-inhibiting antibodies. Together, these results are consistent with a protective role of IFN-γ during the heterologous response against influenza virus independently of the generation and local recruitment of cross-reactive CTLs.     

Human Cytotoxic T Lymphocytes Directed to Seasonal Influenza A Viruses Cross-React with the Newly Emerging H7N9 Virus

In February 2013, zoonotic transmission of a novel influenza A virus of the H7N9 subtype was reported in China. Although at present no sustained human-to-human transmission has been reported, a pandemic outbreak of this H7N9 virus is feared. Since neutralizing antibodies to the hemagglutinin (HA) globular head domain of the virus are virtually absent in the human population, there is interest in identifying other correlates of protection, such as cross-reactive CD8⁺ T cells (cytotoxic T lymphocytes [CTLs]) elicited during seasonal influenza A virus infections. These virus-specific CD8⁺ T cells are known to recognize conserved internal proteins of influenza A viruses predominantly, but it is unknown to what extent they cross-react with the newly emerging H7N9 virus. Here, we assessed the cross-reactivity of seasonal H3N2 and H1N1 and pandemic H1N1 influenza A virus-specific polyclonal CD8⁺ T cells, obtained from HLA-typed study subjects, with the novel H7N9 virus. The cross-reactivity of CD8⁺ T cells to H7N9 variants of known influenza A virus epitopes and H7N9 virus-infected cells was determined by their gamma interferon (IFN-γ) response and lytic activity. It was concluded that, apart from recognition of individual H7N9 variant epitopes, CD8⁺ T cells to seasonal influenza viruses display considerable cross-reactivity with the novel H7N9 virus. The presence of these cross-reactive CD8⁺ T cells may afford some protection against infection with the new virus.     

Overexpression of Interleukin-15 Compromises CD4-Dependent Adaptive Immune Responses against Herpes Simplex Virus 2

Interleukin-15 (IL-15) is necessary for the development and function of NK/NKT cells and the maintenance of naive and memory CD8⁺ T cells. In the absence of IL-15, protective innate immunity is not available; however, a functional adaptive immune response against vaginal herpes simplex virus 2 (HSV-2) is generated. Mice overexpressing IL-15 (IL-15tg mice) have higher numbers of NK cells, greater NK-derived gamma interferon, and more CD8⁺ T cells. Here we examined the consequences of IL-15 overexpression for innate and adaptive immunity against genital HSV-2. Surprisingly, IL-15tg mice immunized against HSV-2 were not protected against genital HSV-2 challenge compared to control immunized mice. IL-15tg mice had a higher frequency of NK cells in the genital mucosa than control mice. However, immunized IL-15tg mice had significantly lower numbers of HSV-2-specific CD4⁺ T cells than B6 mice. We then confirmed that CD4⁺ T cells, but not CD8⁺ T cells, are essential for protection against intravaginal HSV-2 challenge. Since we observed less protection in immunized IL-15tg mice, we then examined if the adaptive immune responses generated in an environment with overexpression of IL-15 could provide protection against HSV-2 in an environment with normal levels of IL-15 expression. We adoptively transferred immunized cells from IL-15tg and B6 mice into naive RAG-1^−/− mice and found that the cells from immunized IL-15tg mice were able to provide protection in this IL-15-normal environment. Our data suggest that overexpression of IL-15 results in a reduced CD4⁺ T cell-mediated adaptive immune response against genital HSV-2.     

Inactivated Simian Immunodeficiency Virus-Pulsed Autologous Fresh Blood Cells as an Immunotherapy Strategy

Practical immunotherapies for human immunodeficiency virus infection are needed. We evaluated inactivated simian immunodeficiency virus (SIV) pulsed onto fresh peripheral blood mononuclear cells in 12 pigtail macaques with chronic SIV_mac251 infection for T-cell immunogenicity in a randomized cross-over design study. The immunotherapy was safe and convincingly induced high levels of SIV-specific CD4⁺ T-cell responses (mean, 5.9% ± 1.3% of all CD4⁺ T cells) and to a lesser extent SIV-specific CD8⁺ T-cell responses (mean, 0.7% ± 0.4%). Responses were primarily directed toward Gag and less frequently toward Env but not Pol or regulatory/accessory SIV proteins. T-cell responses against Gag were generally broad and polyfunctional, with a mean of 2.7 CD4⁺ T-cell epitopes mapped per animal and more than half of the SIV Gag-specific CD4⁺ T cells expressing three or more effector molecules. The immunogenicity was comparable to that found in previous studies of peptide-pulsed blood cells. Despite the high-level immunogenicity, no reduction in viral load was observed in the chronically viremic macaques. This contrasts with our studies of immunization with peptide-pulsed blood cells during early SIV infection in macaques. Future studies of inactivated virus-pulsed blood cell immunotherapy during early infection of patients receiving antiretroviral therapy are warranted.     

Dominant CD8+ T-Lymphocyte Responses Suppress Expansion of Vaccine-Elicited Subdominant T Lymphocytes in Rhesus Monkeys Challenged with Pathogenic Simian-Human Immunodeficiency Virus

Emerging data suggest that a cytotoxic T-lymphocyte response against a diversity of epitopes confers greater protection against a human immunodeficiency virus/simian immunodeficiency virus infection than does a more focused response. To facilitate the creation of vaccine strategies that will generate cellular immune responses with the greatest breadth, it will be important to understand the mechanisms employed by the immune response to regulate the relative magnitudes of dominant and nondominant epitope-specific cellular immune responses. In this study, we generated dominant Gag p11C- and subdominant Env p41A-specific CD8⁺ T-lymphocyte responses in Mamu-A*01⁺ rhesus monkeys through vaccination with plasmid DNA and recombinant adenovirus encoding simian-human immunodeficiency virus (SHIV) proteins. Infection of vaccinated Mamu-A*01⁺ rhesus monkeys with a SHIV Gag Δp11C mutant virus generated a significantly increased expansion of the Env p41A-specific CD8⁺ T-lymphocyte response in the absence of secondary Gag p11C-specific CD8⁺ T-lymphocyte responses. These results indicate that the presence of the Gag p11C-specific CD8⁺ T-lymphocyte response following virus challenge may exert suppressive effects on primed Env p41A-specific CD8⁺ T-lymphocyte responses. These findings suggest that immunodomination exerted by dominant responses during SHIV infection may diminish the breadth of recall responses primed during vaccination.CD8⁺ T lymphocyte responses play a central role in controlling human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) infections in nonhuman primates (18, 20, 29, 41). Naturally occurring virus-specific CD8⁺ T-lymphocyte responses typically focus on a limited number of dominant epitopes (52). However, accumulating data indicate that a broad cellular immune response, in which multiple viral epitopes are recognized by CD8⁺ T lymphocytes, confers better protection against viral replication than a restricted cellular immune response (26, 33). Therefore, it has been suggested that increasing the magnitude of subdominant epitope-specific responses may increase the breadth of a cellular immune response and provide enhanced protection against HIV/SIV replication.An understanding of the factors that influence the immunodominance hierarchy of viral epitopes will be needed to develop vaccination strategies that can generate the greatest breadth of virus-specific CD8⁺ T-lymphocyte responses. Differences in antigen processing, competition between epitope peptides for major histocompatibility complex (MHC) class I molecules, T-cell receptor (TCR) repertoire, TCR affinity for peptide class I complexes, and immunodomination have been shown to contribute to the dominance of an epitope-specific response (6, 10, 24, 32, 45, 52). In addition, studies have shown that immunodominance patterns for T-lymphocyte epitopes may differ following a primary and secondary exposure to the same viral antigen (4, 5, 43).In the present study, we observed that Mamu-A*01⁺ rhesus monkeys primed with plasmid DNA and boosted with recombinant adenovirus (rAd) vaccines encoding SIVmac239 Gag-Pol-Nef and HIV-1 Env proteins generated Gag p11C- and Env p41A-specific CD8⁺ T-lymphocyte responses of comparable magnitude. However, while there was a significant expansion of Gag p11C-specific CD8⁺ T-lymphocyte populations following challenge with pathogenic simian-human immunodeficiency virus 89.6P (SHIV-89.6P), there was no significant expansion of the Env p41A-specific CD8⁺ T-lymphocyte populations. We hypothesized that factors influencing the relative immunodominance of the primed Gag p11C- and Env p41A-specific CD8⁺ T-lymphocyte responses after viral challenge may have contributed to the observed differences in their secondary expansion. In the present study, we sought to identify the potential factors contributing to this immunodominance.     

Protective CD4+ and CD8+ T Cells against Influenza Virus Induced by Vaccination with Nucleoprotein DNA

DNA vaccination is an effective means of eliciting both humoral and cellular immunity, including cytotoxic T lymphocytes (CTL). Using an influenza virus model, we previously demonstrated that injection of DNA encoding influenza virus nucleoprotein (NP) induced major histocompatibility complex class I-restricted CTL and cross-strain protection from lethal virus challenge in mice (J. B. Ulmer et al., Science 259:1745–1749, 1993). In the present study, we have characterized in more detail the cellular immune responses induced by NP DNA, which included robust lymphoproliferation and Th1-type cytokine secretion (high levels of gamma interferon and interleukin-2 [IL-2], with little IL-4 or IL-10) in response to antigen-specific restimulation of splenocytes in vitro. These responses were mediated by CD4⁺ T cells, as shown by in vitro depletion of T-cell subsets. Taken together, these results indicate that immunization with NP DNA primes both cytolytic CD8⁺ T cells and cytokine-secreting CD4⁺ T cells. Further, we demonstrate by adoptive transfer and in vivo depletion of T-cell subsets that both of these types of T cells act as effectors in protective immunity against influenza virus challenge conferred by NP DNA.Cellular immune responses play an important role in protection from disease caused by infectious pathogens, such as viruses and certain bacteria (e.g., Mycobacterium tuberculosis). The specific T cells involved in conferring immunity can include both CD4⁺ and CD8⁺ T cells, often through the action of secreted cytokines and cytolytic activity, respectively. Certain types of vaccines, such as subunit proteins and whole or partially purified preparations of inactivated organisms, in general induce CD4⁺ T-cell responses but not CD8⁺ cytotoxic T lymphocytes (CTL). In contrast, live attenuated organisms and subunit proteins formulated with certain experimental adjuvants can induce both types of responses. Recently, a different approach consisting of direct immunization with plasmid DNA expression vectors (i.e., DNA vaccines) has shown promise as a viable means of inducing broad-spectrum T-cell responses. The effectiveness of DNA vaccines in animal models is likely due, at least in part, to expression of antigens in situ (35), leading to the induction of CTL (29), antibodies (3, 4, 10, 21, 22, 32), and cytokine-secreting lymphocyte responses (12, 36). During the past 5 years, many reports have been published on the immunogenicity of DNA vaccines encoding various antigens in several animal models, thereby illustrating the applicability of the technology to many pathogens (for a review, see reference 6). However, in only a few instances has the nature of the effector cells responsible for protective immunity been described (7, 16). In the present study, we have analyzed in detail the cellular immune responses induced by influenza virus nucleoprotein (NP) DNA and have established that both CD4⁺ T cells secreting Th1-type cytokines and CD8⁺ cytotoxic T cells play important effector roles in heterosubtypic protective immunity against lethal influenza virus challenge in mice.     

Memory CD4+ T-cell-mediated protection from lethal coronavirus encephalomyelitis

The antiviral role of CD4⁺ T cells in virus-induced pathologies of the central nervous system (CNS) has not been explored extensively. Control of neurotropic mouse hepatitis virus (JHMV) requires the collaboration of CD4⁺ and CD8⁺ T cells, with CD8⁺ T cells providing direct perforin and gamma interferon (IFN-γ)-mediated antiviral activity. To distinguish bystander from direct antiviral contributions of CD4⁺ T cells in virus clearance and pathology, memory CD4⁺ T cells purified from wild type (wt), perforin-deficient (PKO), and IFN-γ-deficient (GKO) immune donors were transferred to immunodeficient SCID mice prior to CNS challenge. All three donor CD4⁺ T-cell populations controlled CNS virus replication at 8 days postinfection, indicating IFN-γ- and perforin-independent antiviral function. Recipients of GKO CD4⁺ T cells succumbed more rapidly to fatal disease than untreated control infected mice. In contrast, wt and PKO donor CD4⁺ T cells cleared infectious virus to undetectable levels and protected from fatal disease. Recipients of all CD4⁺ T-cell populations exhibited demyelination. However, it was more severe in wt CD4⁺ T-cell recipients. These data support a role of CD4⁺ T cells in virus clearance and demyelination. Despite substantial IFN-γ-independent antiviral activity, IFN-γ was crucial in providing protection from death. IFN-γ reduced neutrophil accumulation and directed macrophages to white matter but did not ameliorate myelin loss.     

T lymphocytes contribute to antiviral immunity and pathogenesis in experimental human metapneumovirus infection

Human metapneumovirus (hMPV), a member of the family Paramyxoviridae, is a leading cause of lower respiratory tract infections in children, the elderly, and immunocompromised patients. Virus- and host-specific mechanisms of pathogenesis and immune protection are not fully understood. By an intranasal inoculation model, we show that hMPV-infected BALB/c mice developed clinical disease, including airway obstruction and hyperresponsiveness (AHR), along with histopathologic evidence of lung inflammation and viral replication. hMPV infection protected mice against subsequent viral challenge, as demonstrated by undetectable viral titers, lack of body weight loss, and a significant reduction in the level of lung inflammation. No cross-protection with other paramyxoviruses, such as respiratory syncytial virus, was observed. T-lymphocyte depletion studies showed that CD4⁺ and CD8⁺ T cells cooperate synergistically in hMPV eradication during primary infection, but CD4⁺ more than CD8⁺ T cells also enhanced clinical disease and lung pathology. Concurrent depletion of CD4⁺ and CD8⁺ T cells completely blocked airway obstruction as well as AHR. Despite impaired generation of neutralizing anti-hMPV antibodies in the absence of CD4⁺ T cells, mice had undetectable viral replication after hMPV challenge and were protected from clinical disease, suggesting that protection can be provided by an intact CD8⁺ T-cell compartment. Whether these findings have implications for naturally acquired human infections remains to be determined.     

Influenza Virus-Infected Epithelial Cells Present Viral Antigens to Antigen-Specific CD8+ Cytotoxic T Lymphocytes

We have investigated the mechanisms involved in the clearance of viral infection at the epithelium level by analyzing the activity of influenza virus-specific cytotoxic T lymphocytes (CTL) against virus-infected CMT-93 intestinal epithelial cells. Epithelial cells infected with live influenza virus effectively present viral antigens and were lysed by both homotypic and heterotypic influenza virus-specific CD8⁺ T cells. These results shed new light on the control of viral infection through the elimination of virus-infected epithelial cells by virus-specific CTL and demonstrate that CMT-93 cells furnish an appropriate model for in vitro evaluation of CTL activity against virus-infected epithelial cells.