Quantifying antigen-specific CD4 T cells during a viral infection: CD4 T cell responses are larger than we think

The number of virus-specific CD8 T cells increases substantially during an acute infection. Up to 90% of CD8 T cells are virus specific following lymphocytic choriomeningitis virus (LCMV) infection. In contrast, studies identifying virus-specific CD4 T cell epitopes have indicated that CD4 T cells often recognize a broader array of Ags than CD8 T cells, consequently making it difficult to accurately quantify the total magnitude of pathogen-specific CD4 T cell responses. In this study, we show that CD4 T cells become CD11a(hi)CD49d(+) after LCMV infection and retain this expression pattern into memory. During the effector phase, all the LCMV-specific IFN-γ(+) CD4 T cells display a CD11a(hi)CD49d(+) cell surface expression phenotype. In addition, only memory CD11a(hi)CD49d(+) CD4 T cells make IFN-γ after stimulation. Furthermore, upon secondary LCMV challenge, only CD11a(hi)CD49d(+) memory CD4 T cells from LCMV-immune mice undergo proliferative expansion, demonstrating that CD11a(hi)CD49d(+) CD4 T cells are truly Ag specific. Using the combination of CD11a and CD49d, we demonstrate that up to 50% of the CD4 T cells are virus specific during the peak of the LCMV response. Our results indicate that the magnitude of the virus-specific CD4 T cell response is much greater than previously recognized.     

Direct ex vivo kinetic and phenotypic analyses of CD8(+) T-cell responses induced by DNA immunization

CD8(+) T-cell responses can be induced by DNA immunization, but little is known about the kinetics of these responses in vivo in the absence of restimulation or how soon protective immunity is conferred by a DNA vaccine. It is also unclear if CD8(+) T cells primed by DNA vaccines express the vigorous effector functions characteristic of cells primed by natural infection or by immunization with a recombinant live virus vaccine. To address these issues, we have used the sensitive technique of intracellular cytokine staining to carry out direct ex vivo kinetic and phenotypic analyses of antigen-specific CD8(+) T cells present in the spleens of mice at various times after (i) a single intramuscular administration of a plasmid expressing the nucleoprotein (NP) gene from lymphocytic choriomeningitis virus (LCMV), (ii) infection by a recombinant vaccinia virus carrying the same protein (vvNP), or (iii) LCMV infection. In addition, we have evaluated the rapidity with which protective immunity against both lethal and sublethal LCMV infections is achieved following DNA vaccination. The CD8(+) T-cell response in DNA-vaccinated mice was slightly delayed compared to LCMV or vvNP vaccinees, peaking at 15 days postimmunization. Interestingly, the percentage of antigen-specific CD8(+) T cells present in the spleen at day 15 and later time points was similar to that observed following vvNP infection. T cells primed by DNA vaccination or by infection exhibited similar cytokine expression profiles and had similar avidities for an immunodominant cytotoxic T lymphocyte epitope peptide, implying that the responses induced by DNA vaccination differ quantitatively but not qualitatively from those induced by live virus infection. Surprisingly, protection from both lethal and sublethal LCMV infections was conferred within 1 week of DNA vaccination, well before the peak of the CD8(+) T-cell response.     

High frequency of virus-specific interleukin-2-producing CD4(+) T cells and Th1 dominance during lymphocytic choriomeningitis virus infection

Analysis of C57BL/6 mice acutely infected with lymphocytic choriomeningitis virus (LCMV) by using intracellular cytokine staining revealed a high frequency (2 to 10%) of CD4(+) T cells secreting the Th1-associated cytokines interleukin-2 (IL-2), gamma interferon (IFN-gamma), and tumor necrosis factor alpha, with no concomitant increase in the frequency of CD4(+) T cells secreting the Th2-associated cytokines IL-4, IL-5, and IL-10 following stimulation with viral peptides. In LCMV-infected C57BL/6 CD8(-/-) mice, more than 20% of the CD4(+) T cells secreted IFN-gamma after viral peptide stimulation, whereas less than 1% of the CD4(+) T cells secreted IL-4 under these same conditions. Mice persistently infected with a high dose of LCMV clone 13 also generated a virtually exclusive Th1 response. Thus, LCMV induces a much more profound virus-specific CD4(+) T-cell response than previously recognized, and it is dramatically skewed to a Th1 phenotype.     

Critical Role for Perforin-, Fas/FasL-, and TNFR1-Mediated Cytotoxic Pathways in Down-Regulation of Antigen-Specific T Cells during Persistent Viral Infection

Viral persistence following infection with invasive strains of lymphocytic choriomeningitis virus (LCMV) can be achieved by selective down-regulation of virus-specific T lymphocytes. High viral burden in the onset of infection drives responding cells into functional unresponsiveness (anergy) that can be followed by their physical elimination. In this report, we studied down-regulation of the virus-specific CD8(+)-T-cell response during persistent infection of adult mice with LCMV, with emphasis on the role of perforin-, Fas/FasL-, or tumor necrosis factor receptor 1 (TNFR1)-mediated cytolysis in regulating T-cell homeostasis. The results reveal that the absence of perforin, Fas-ligand, or TNFR1 has no significant effect on the kinetics of proliferation and functional inactivation of virus-specific CD8(+) T cells in the onset of chronic LCMV infection. However, these molecules play a critical role in the homeostatic regulation of T cells, influencing the longevity of the virus-specific CD8(+)-T-cell population once it has become anergic. Thus, CD8(+) T cells specific to the dominant LCMV NP(396-404) epitope persist in an anergic state for at least 70 days in perforin-, FasL-, or TNFR1-deficient mice, but they were eliminated by day 30 in C57BL/6 controls. These effects were additive as shown by a deficit of apoptotic death of NP(396-404) peptide-specific CD8(+) T cells in mice lacking both perforin and TNFR1. This suggests a role for perforin-, FasL-, and TNFR1-mediated pathways in down-regulation of the antiviral T cell response during persistent viral infection by determining the fate of antigen-specific T cells. Moreover, virus-specific anergic CD8(+) T cells in persistently infected C57BL/6 mice contain higher levels of Bcl-2 and Bcl-XL than functionally intact T cells generated during acute LCMV infection. In the case of proapoptotic factors, Bax expression did not differ between T-cell populations and Bad was below the limit of detection in all samples. As expression of the Bcl-2 family members controls susceptibility to apoptosis, this finding may provide a molecular basis for the survival of anergic cells under conditions of prolonged antigen stimulation.     

In vivo selection of a lymphocytic choriomeningitis virus variant that affects recognition of the GP33-43 epitope by H-2Db but not H-2Kb

CD8 T cells drive the protective immune response to lymphocytic choriomeningitis virus (LCMV) infection and are thus a determining force in the selection of viral variants. To examine how escape mutations affect the presentation and recognition of overlapping T-cell epitopes, we isolated an LCMV variant that is not recognized by T-cell receptor (TCR)-transgenic H-2Db-restricted LCMV GP33-41-specific cytotoxic T lymphocytes (CTL). The variant virus carried a single-amino-acid substitution (valine to alanine) at position 35 of the viral glycoprotein. This region of the LCMV glycoprotein encodes both the Db-restricted GP33-43 epitope and a second epitope (GP34-42) presented by the Kb molecule. We determined that the V-to-A CTL escape mutant failed to induce a Db GP33-43-specific CTL response and that Db-restricted GP33-43-specific CTL induced by the wild-type LCMV strain were unable to kill target cells infected with the variant LCMV strain. In contrast, the Kb-restricted response was much less affected. We found that the V-to-A substitution severely impaired peptide binding to Db but not to Kb molecules. Strikingly, the V-to-A mutation did not change any of the anchor residues, and the dramatic effect on binding was therefore unexpected. The strong decrease in Db binding explains why the variant virus escapes the Db GP33-43-specific response but still elicits the Kb-restricted response. These findings also illustrate that mutations within regions encoding overlapping T-cell epitopes can differentially affect the presentation and recognition of individual epitopes.     

The CD8+ T-cell response to lymphocytic choriomeningitis virus involves the L antigen: uncovering new tricks for an old virus

CD8(+) T-cell responses control lymphocytic choriomeningitis virus (LCMV) infection in H-2(b) mice. Although antigen-specific responses against LCMV infection are well studied, we found that a significant fraction of the CD8(+) CD44(hi) T-cell response to LCMV in H-2(b) mice was not accounted for by known epitopes. We screened peptides predicted to bind major histocompatibility complex class I and overlapping 15-mer peptides spanning the complete LCMV proteome for gamma interferon (IFN-gamma) induction from CD8(+) T cells derived from LCMV-infected H-2(b) mice. We identified 19 novel epitopes. Together with the 9 previously known, these epitopes account for the total CD8(+) CD44(hi) response. Thus, bystander T-cell activation does not contribute appreciably to the CD8(+) CD44(hi) pool. Strikingly, 15 of the 19 new epitopes were derived from the viral L polymerase, which, until now, was not recognized as a target of the cellular response induced by LCMV infection. The L epitopes induced significant levels of in vivo cytotoxicity and conferred protection against LCMV challenge. Interestingly, protection from viral challenge was best correlated with the cytolytic potential of CD8(+) T cells, whereas IFN-gamma production and peptide avidity appear to play a lesser role. Taken together, these findings illustrate that the LCMV-specific CD8(+) T-cell response is more complex than previously appreciated.     

Extralymphatic virus sanctuaries as a consequence of potent T-cell activation

T helper cells can support the functions of CD8(+) T cells against persistently infecting viruses such as murine lymphocytic choriomeningitis virus (LCMV), cytomegalovirus, hepatitis C virus and HIV. These viruses often resist complete elimination and remain detectable at sanctuary sites, such as the kidneys and other extralymphatic organs. The mechanisms underlying this persistence are not well understood. Here we show that mice with potent virus-specific T-cell responses have reduced levels and delayed formation of neutralizing antibodies, and these mice fail to clear LCMV from extralymphatic epithelia. Transfer of virus-specific B cells but not virus-specific T cells augmented virus clearance from persistent sites. Virus elimination from the kidneys was associated with the formation of IgG deposits in the interstitial space, presumably from kidney-infiltrating B cells. CD8(+) T cells in the kidneys of mice that did not clear virus from this site were activated but showed evidence of exhaustion. Thus, we conclude that in this model of infection, site-specific virus persistence develops as a consequence of potent immune activation coupled with reductions in virus-specific neutralizing antibodies. Our results suggest that sanctuary-site formation depends both on organ anatomy and on the induction of different adaptive immune effector mechanisms. Boosting T-cell responses alone may not reduce virus persistence.     

Intranasal delivery of recombinant parvovirus-like particles elicits cytotoxic T-cell and neutralizing antibody responses

We previously demonstrated that chimeric porcine parvovirus-like particles (PPV:VLP) carrying heterologous epitopes, when injected intraperitoneally into mice without adjuvant, activate strong CD4(+) and CD8(+) T-cell responses specific for the foreign epitopes. In the present study, we investigated the immunogenicity of PPV:VLP carrying a CD8(+) T-cell epitope from the lymphocytic choriomeningitis virus (LCMV) administered by mucosal routes. Mice immunized intranasally with recombinant PPV:VLP, in the absence of adjuvant, developed high levels of PPV-specific immunoglobulin G (IgG) and/or IgA in their serum, as well as in mucosal sites such as the bronchoalveolar and intestinal fluids. Antibodies in sera from mice immunized parenterally or intranasally with PPV:VLP were strongly neutralizing in vitro. Intranasal immunization with PPV:VLP carrying the LCMV CD8(+) T-cell epitope also elicited a strong peptide-specific cytotoxic-T-cell (CTL) response. In contrast, mice orally immunized with recombinant PPV:VLP did not develop any antibody or CTL responses. We also showed that mice primed with PPV:VLP are still able to develop strong CTL responses after subsequent immunization with chimeric PPV:VLP carrying a foreign CD8(+) T-cell epitope. These results highlight the attractive potential of PPV:VLP as a safe, nonreplicating antigen carrier to stimulate systemic and mucosal immunity after nasal administration.     

Naive precursor frequencies and MHC binding rather than the degree of epitope diversity shape CD8+ T cell immunodominance

The primary CD8(+) T cell response of C57BL/6J mice against the 28 known epitopes of lymphocytic choriomeningitis virus (LCMV) is associated with a clear immunodominance hierarchy whose mechanism has yet to be defined. To evaluate the role of epitope competition in immunodominance, we manipulated the number of CD8(+) T cell epitopes that could be recognized during LCMV infection. Decreasing epitope numbers, using a viral variant lacking dominant epitopes or C57BL/6J mice lacking H-2K(b), resulted in minor response increases for the remaining epitopes and no new epitopes being recognized. Increasing epitope numbers by using F(1) hybrid mice, delivery by recombinant vaccinia virus, or epitope delivery as a pool in IFA maintained the overall response pattern; however, changes in the hierarchy did become apparent. MHC binding affinity of these epitopes was measured and was found to not strictly predict the hierarchy since in several cases similarly high binding affinities were associated with differences in immunodominance. In these instances the naive CD8(+) T cell precursor frequency, directly measured by tetramer staining, correlated with the response hierarchy seen after LCMV infection. Finally, we investigated an escape mutant of the dominant GP33-41 epitope that elicited a weak response following LCMV variant virus infection. Strikingly, dominance loss likely reflects a substantial reduction in frequencies of naive precursors specific for this epitope. Thus, our results indicate that an intrinsic property of the epitope (MHC binding affinity) and an intrinsic property of the host (naive precursor frequency) jointly dictate the immunodominance hierarchy of CD8(+) T cell responses.     

Cross-protection against lymphocytic choriomeningitis virus mediated by a CD4+ T-cell clone specific for an envelope glycoprotein epitope of Lassa virus.

Recombinant vaccinia virus expressing the Lassa virus (LV) envelope glycoprotein precursor, V-LSGPC, was used to study the basis of LV-induced cross-protective immunity against the closely related arenavirus lymphocytic choriomeningitis virus (LCMV). C3H/HeJ mice primed with V-LSGPC developed neither circulating antibodies nor CD8+ cytotoxic T cells specific for LCMV, yet they resisted a normally lethal LCMV challenge. Spleen cells from such mice gave a proliferative response to LCMV in vitro that was inhibitable by anti-CD4 antibody. Synthetic peptides corresponding to predicted T-cell sites common to the envelope glycoprotein precursor (GP-C) of LV and that of LCMV were used to map the specificity of the proliferative response to an epitope located between amino acids 403 and 417 of LV GP-C. Several CD4+ T-cell clones specific for the 403-417 peptide were isolated and found to produce gamma interferon in response to both the peptide and LCMV. One of these clones, C9, was selected for further study. C9 lysed I-AK-bearing target cells, and when adoptively transferred to C3H/HeJ mice, it was capable of mediating both a peptide-specific delayed hypersensitivity reaction and resistance to lethal LCMV challenge. These collective findings demonstrate, for the first time, that CD4+ T cells can play a major role in arenavirus-specific cross-protective immunity.     

In vivo induction of a high-avidity, high-frequency cytotoxic T-lymphocyte response is associated with antiviral protective immunity

Many approaches are currently being developed to deliver exogenous antigen into the major histocompatibility complex class I-restricted antigen pathway, leading to in vivo priming of CD8(+) cytotoxic T cells. One attractive possibility consists of targeting the antigen to phagocytic or macropinocytic antigen-presenting cells. In this study, we demonstrate that strong CD8(+) class I-restricted cytotoxic responses are induced upon intraperitoneal immunization of mice with different peptides, characterized as CD8(+) T-cell epitopes, bound to 1-microm synthetic latex microspheres and injected in the absence of adjuvant. The cytotoxic response induced against a lymphocytic choriomeningitis virus (LCMV) peptide linked to these microspheres was compared to the cytotoxic T-lymphocyte (CTL) response obtained upon immunization with the nonreplicative porcine parvovirus-like particles (PPV:VLP) carrying the same peptide (PPV:VLP-LCMV) previously described (C. Sedlik, M. F. Saron, J. Sarraseca, I. Casal, and C. Leclerc, Proc. Natl. Acad. Sci. USA 94:7503-7508, 1997). We show that the induction of specific CTL activity by peptides bound to microspheres requires CD4(+) T-cell help in contrast to the CTL response obtained with the peptide delivered by viral pseudoparticles. Furthermore, PPV:VLP are 100-fold more efficient than microspheres in generating a strong CTL response characterized by a high frequency of specific T cells of high avidity. Moreover, PPV:VLP-LCMV are able to protect mice against a lethal LCMV challenge whereas microspheres carrying the LCMV epitope fail to confer such protection. This study demonstrates the crucial involvement of the frequency and avidity of CTLs in conferring antiviral protective immunity and highlights the importance of considering these parameters when developing new vaccine strategies.     

Critical role for alpha/beta and gamma interferons in persistence of lymphocytic choriomeningitis virus by clonal exhaustion of cytotoxic T cells

Under conditions of high antigenic load during infection with invasive lymphocytic choriomeningitis virus (LCMV) strains, virus can persist by selective clonal exhaustion of antigen-specific CD8(+) T cells. In this work we studied the down-regulation of the virus-specific CD8(+)-T-cell response during a persistent infection of adult mice, with particular emphasis on the contribution of the interferon response in promoting host defense. Studies were conducted by infecting mice deficient in receptors for type I (alpha/beta interferon [IFN-alpha/beta]), type II (IFN-gamma), and both type I and II IFNs with LCMV isolates that vary in their capacity to induce T-cell exhaustion. The main conclusions of this study are as follows. (i) IFNs play a critical role in LCMV infection by reducing viral loads in the initial stages of infection and thus modifying both the extent of CD8(+)-T-cell exhaustion and the course of infection. The importance of IFNs in this context varies with the biological properties of the LCMV strain. (ii) An inverse correlation exists between antigen persistence and responsiveness of virus-specific CD8(+) T cells. This results in distinct programs of activation or tolerance (functional unresponsiveness and/or physical elimination of antigen-specific cells) during acute and chronic virus infections, respectively. (iii) A successful immune response associated with definitive viral clearance requires an appropriate balance between cellular and humoral components of the immune system. We discuss the role of IFNs in influencing virus-specific T cells that determine the outcome of persistent infections.     

Low CD8 T-cell proliferative potential and high viral load limit the effectiveness of therapeutic vaccination

Therapeutic vaccination has the potential to boost immune responses and enhance viral control during chronic infections. However, many therapeutic vaccination approaches have fallen short of expectations, and effective boosting of antiviral T-cell responses is not always observed. To examine these issues, we studied the impact of therapeutic vaccination, using a murine model of chronic infection with lymphocytic choriomeningitis virus (LCMV). Our results demonstrate that therapeutic vaccination using a recombinant vaccinia virus expressing the LCMV GP33 CD8 T-cell epitope can be effective at accelerating viral control. However, mice with lower viral loads at the time of vaccination responded better to therapeutic vaccination than did those with high viral loads. Also, the proliferative potential of GP33-specific CD8 T cells from chronically infected mice was substantially lower than that of GP33-specific memory CD8 T cells from mice with immunity to LCMV, suggesting that poor T-cell expansion may be an important reason for suboptimal responses to therapeutic vaccination. Thus, our results highlight the potential positive effects of therapeutic vaccination on viral control during chronic infection but also provide evidence that a high viral load at the time of vaccination and the low proliferative potential of responding T cells are likely to limit the effectiveness of therapeutic vaccination.     

Independent regulation of lymphocytic choriomeningitis virus-specific T cell memory pools: relative stability of CD4 memory under conditions of CD8 memory T cell loss

Infection of mice with a series of heterologous viruses causes a reduction of memory CD8(+) T cells specific to viruses from earlier infections, but the fate of the virus-specific memory CD4(+) T cell pool following multiple virus infections has been unknown. We have previously reported that the virus-specific CD4(+) Th precursor (Thp) frequency remains stable into long-term immunity following lymphocytic choriomeningitis virus (LCMV) infection. In this study, we questioned whether heterologous virus infections or injection with soluble protein CD4 Ags would impact this stable LCMV-specific CD4(+) Thp memory pool. Limiting dilution analyses for IL-2-producing cells and intracellular cytokine staining for IFN-gamma revealed that the LCMV-specific CD4(+) Thp frequency remains relatively stable following multiple heterologous virus infections or protein Ag immunizations, even under conditions that dramatically reduce the LCMV-specific CD8(+) CTL precursor frequency. These data indicate that the CD4(+) and CD8(+) memory T cell pools are regulated independently and that the loss in CD8(+) T cell memory following heterologous virus infections is not a consequence of a parallel loss in the memory CD4(+) T cell population.     

Targeting of Non-Dominant Antigens as a Vaccine Strategy to Broaden T-Cell Responses during Chronic Viral Infection

In this study, we compared adenoviral vaccine vectors with the capacity to induce equally potent immune responses against non-dominant and immunodominant epitopes of murine lymphocytic choriomeningitis virus (LCMV). Our results demonstrate that vaccination targeting non-dominant epitopes facilitates potent virus-induced T-cell responses against immunodominant epitopes during subsequent challenge with highly invasive virus. In contrast, when an immunodominant epitope was included in the vaccine, the T-cell response associated with viral challenge remained focussed on that epitope. Early after challenge with live virus, the CD8+ T cells specific for vaccine-encoded epitopes, displayed a phenotype typically associated with prolonged/persistent antigenic stimulation marked by high levels of KLRG-1, as compared to T cells reacting to epitopes not included in the vaccine. Notably, this association was lost over time in T cells specific for the dominant T cell epitopes, and these cells were fully capable of expanding in response to a new viral challenge. Overall, our data suggests a potential for broadening of the antiviral CD8+ T-cell response by selecting non-dominant antigens to be targeted by vaccination. In addition, our findings suggest that prior adenoviral vaccination is not likely to negatively impact the long-term and protective immune response induced and maintained by a vaccine-attenuated chronic viral infection.     

Quantification of epitope-specific MHC class-II-restricted T cells following lymphocytic choriomeningitis virus infection

CD4(+) T cells are critical for the control of many viruses; however, the numbers of virus-specific CD4(+) cells that are expanded following infection are unknown. We have addressed this issue by enumerating virus-specific, MHC class-II-restricted T cells following infection of mice with lymphocytic choriomeningitis virus (LCMV). We have found that the numbers of T cells that produce interferon-gamma in response to stimulation with three different class-II-restricted LCMV epitopes increase from undetectable numbers in noninfected animals to between 4 x 10(5) and 2 x 10(6) cells per spleen at the peak of the T cell response. This contrasts with the numbers of virus-specific class-I-restricted T cells which expand to 1 x 10(7) to 2 x 10(7) cells per spleen during the same time period. We could not reproducibly detect virus-specific class-I-restricted or class-II-restricted T cells that produced interleukin-4 at any time following LCMV infection, indicating that infection with this virus induces a predominantly type 1 cytokine response. In contrast to the rapid decrease in the numbers of class-I-restricted T cells, the numbers of LCMV-specific class-II-restricted T cells declined gradually following the peak of the T cell response. We demonstrate, therefore, that following infection with LCMV there is expansion of both class-I-restricted and class-II-restricted virus-specific T cells; however, the degree of expansion of class-II-restricted T cells is substantially less than that observed for class-I-restricted cells. Furthermore, the downregulation phase of the class-II-restricted response is protracted compared with the precipitous contraction of the antiviral CD8(+) T cell response.     

Endosomal/lysosomal targeting of a single helper T-cell epitope of an intracellular bacterium by DNA immunisation induces a specific T-cell subset and partial protective immunity in vivo

We evaluated here the effect of the intracellular targeting of a helper T-cell (Th) epitope, literiolysin O 215-226 derived from Listeria monocytogenes, on induction of a specific Th by gene gun immunisation. Immunisation of C3H/He mice with pE215LAMP plasmid encoding the Th epitope fused with the endosomal/lysosomal targeting signal of lysosome-associated membrane protein (LAMP)-1 gave the epitope-specific proliferative responses of CD4(+) T lymphocytes. In addition, specific interferon-gamma production from the splenocytes was observed. Concomitantly, pE215LAMP-immunised mice showed moderate, but significant protective immunity against listerial challenge. These results suggest that the intracellular targeting of a Th epitope to endosomal/lysosomal compartments by DNA immunisation is useful for eliciting a specific Th subset in vivo.     

Development of a new hydrogen peroxide–based vaccine platform

Safe and effective vaccines are crucial for maintaining public health and reducing the global burden of infectious disease. Here we introduce a new vaccine platform that uses hydrogen peroxide (H(2)O(2)) to inactivate viruses for vaccine production. H(2)O(2) rapidly inactivates both RNA and DNA viruses with minimal damage to antigenic structure or immunogenicity and is a highly effective method when compared with conventional vaccine inactivation approaches such as formaldehyde or β-propiolactone. Mice immunized with H(2)O(2)-inactivated lymphocytic choriomeningitis virus (LCMV) generated cytolytic, multifunctional virus-specific CD8(+) T cells that conferred protection against chronic LCMV infection. Likewise, mice vaccinated with H(2)O(2)-inactivated vaccinia virus or H(2)O(2)-inactivated West Nile virus showed high virus-specific neutralizing antibody titers and were fully protected against lethal challenge. Together, these studies demonstrate that H(2)O(2)-based vaccines are highly immunogenic, provide protection against a range of viral pathogens in mice and represent a promising new approach to future vaccine development.     

Dissection of antiviral and immune regulatory functions of tumor necrosis factor receptors in a chronic lymphocytic choriomeningitis virus infection

The effector function of CD8 T cells is mediated via cell-mediated cytotoxicity and production of cytokines like gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). While the roles of perforin-dependent cytotoxicity, IFN-gamma, and TNF-alpha in controlling acute viral infections are well studied, their relative importance in defense against chronic viral infections is not well understood. Using mice deficient for TNF receptor (TNFR) I and/or II, we show that TNF-TNFR interactions have a dual role in mediating viral clearance and downregulating CD8 and CD4 T-cell responses during a chronic lymphocytic choriomeningitis virus (LCMV) infection. While wild-type (+/+) and TNFR II-deficient (p75(-/-)) mice cleared LCMV from the liver and lung, mice deficient in TNFR I (p55(-/-)) or both TNFR I and TNFR II (double knockout [DKO]) exhibited impaired viral clearance. The inability of p55(-/-) and DKO mice to clear LCMV was not a sequel to either suboptimal activation of virus-specific CD8 or CD4 T cells or impairment in trafficking of LCMV-specific CD8 T cells to the liver and lung. In fact, the expansion of LCMV-specific CD8 and CD4 T cells was significantly higher in DKO mice compared to that in +/+, p55(-/-), and p75(-/-) mice. TNFR deficiency did not preclude the physical deletion of CD8 T cells specific for nucleoprotein 396 to 404 but delayed the contraction of CD8 T-cell responses to the epitopes GP33-41 and GP276-285 in the viral glycoprotein. The antibody response to LCMV was not significantly altered by TNFR deficiency. Taken together, these findings have implications in development of immunotherapy in chronic viral infections of humans.     

Molecular and functional dissection of the H-2Db-restricted subdominant cytotoxic T-cell response to lymphocytic choriomeningitis virus

Infection of H-2b mice with lymphocytic choriomeningitis virus (LCMV) generates an H-2Db-restricted cytotoxic T-lymphocyte (CTL) response whose subdominant component is directed against the GP92-101 (CSANNSHHYI) epitope. The aim of this study was to identify the functional parameters accounting for this subdominance. We found that the two naturally occurring (genetically encoded and posttranslationally modified) forms of LCMV GP92-101 were immunogenic, did not act as T-cell antagonists, and bound efficiently to but were unable to form stable complexes with H-2Db, a crucial factor for immunodominance. Thus, the H-2Db-restricted subdominant CTL response to LCMV resulted not from altered T-cell activation but from impaired major histocompatibility complex presentation properties.