Contributions of antinucleoprotein IgG to heterosubtypic immunity against influenza virus

Influenza A virus causes recurring seasonal epidemics and occasional influenza pandemics. Because of changes in envelope glycoprotein Ags, neutralizing Abs induced by inactivated vaccines provide limited cross-protection against new viral serotypes. However, prior influenza infection induces heterosubtypic immunity that accelerates viral clearance of a second strain, even if the external proteins are distinct. In mice, cross-protection can also be elicited by systemic immunization with the highly conserved internal nucleoprotein (NP). Both T lymphocytes and Ab contribute to such cross-protection. In this paper, we demonstrate that anti-NP IgG specifically promoted influenza virus clearance in mice by using a mechanism involving both FcRs and CD8(+) cells. Furthermore, anti-NP IgG rescued poor heterosubtypic immunity in B cell-deficient mice, correlating with enhanced NP-specific CD8 T cell responses. Thus, Ab against this conserved Ag has potent antiviral activity both in naive and in influenza-immune subjects. Such antiviral activity was not seen when mice were vaccinated with another internal influenza protein, nonstructural 1. The high conservation of NP Ag and the known longevity of Ab responses suggest that anti-NP IgG may provide a critically needed component of a universal influenza vaccine.     

A simple mathematical model helps to explain the immunodominance of CD8 T cells in influenza A virus infections

Understanding immunodominance, the phenomenon of epitope-specific T cells expanding in an often distinctly hierarchical fashion, is important for the design of T-cell-based intervention strategies. Several recent studies have investigated immunodominance of H-2D(b)-restricted CD8(+) T cells specific for the nucleoprotein NP366 and acid polymerase PA224 epitopes during influenza A virus infection of C57BL/6 mice. CD8(+) T cells specific for these two epitopes are codominant during primary infection; NP366 dominates during secondary infection. While a number of explanations for this observation have been proposed, none of them can fully account for all the observed data. In this article, we use a simple mathematical model to explain the seemingly inconsistent data. We show that the dynamic interactions between CD8(+) T cells and antigen presentation lead to a situation where CD8(+) T cells are limiting during the initial response whereas antigen is limiting in the secondary response. This "numbers game" between antigen and CD8(+) T cells can reproduce the observed immunodominance of the NP336- and PA224-specific CD8(+) T cells, thereby explaining the reported experimental data.     

Concurrent naive and memory CD8(+) T cell responses to an influenza A virus

Memory Thy-1(+)CD8(+) T cells specific for the influenza A virus nucleoprotein (NP(366-374)) peptide were sorted after staining with the D(b)NP(366) tetramer, labeled with CFSE, and transferred into normal Thy-1.2(+) recipients. The donor D(b)NP(366)(+) T cells recovered 2 days later from the spleens of the Thy-1.2(+) hosts showed the CD62L(low)CD44(high)CD69(low) phenotype, characteristic of the population analyzed before transfer, and were present at frequencies equivalent to those detected previously in mice primed once by a single exposure to an influenza A virus. Analysis of CFSE-staining profiles established that resting tetramer(+) T cells divided slowly over the next 30 days, while the numbers in the spleen decreased about 3-fold. Intranasal infection shortly after cell transfer with a noncross-reactive influenza B virus induced some of the donor D(b)NP(366)(+) T cells to cycle, but there was no increase in the total number of transferred cells. By contrast, comparable challenge with an influenza A virus caused substantial clonal expansion, and loss of the CFSE label. Unexpectedly, the recruitment of naive Thy-1.2(+)CD8(+)D(b)NP(366)(+) host D(b)NP(366)(+) T cells following influenza A challenge was not obviously diminished by the presence of the memory Thy-1.1(+)CD8(+)D(b)NP(366)(+) donor D(b)NP(366)(+) set. Furthermore, the splenic response to an epitope (D(b)PA(224)) derived from the influenza acid polymerase (PA(224-233)) was significantly enhanced in the mice given the donor D(b)NP(366)(+) memory population. These experiments indicate that an apparent recall response may be comprised of both naive and memory CD8(+) T cells.     

Selection of the same major T cell determinants of influenza nucleoprotein after vaccination or exposure to infectious virus.

In the present study, we have compared the T cell antigenic determinants on nucleoprotein (NP) of influenza A/NT/60/68 virus recognized by BALB/c mice (H-2d) after vaccination using several different vehicles with the determinants recognized after exposure to infectious virus. Mice were immunized s.c. with: 1) purified recombinant NP with three different adjuvants--alum, saponin, or CFA; 2) whole inactivated A/Okuda virus in PBS or saponin; or 3) live attenuated Salmonella typhimurium AroA- vector expressing NP. A series of overlapping synthetic peptides that cover more than 90% of the amino acid sequence of NP were used to map the Th cell epitopes. The results showed that the same limited number of major epitopes were recognized after each of the different immunization regimes. Secondary in vivo boosting using the same vehicles as for the primary immunization did not increase the number of different T cell sites recognized. The T cell responses after intranasal infection with infectious A/NT/60/68 or A/PR/8/34 virus also showed a similar pattern of recognition of the major CD4-positive T cell epitopes. The only exception was that the region corresponding to residues 401-419 was only recognized after exposure to NP from A/NT/60/68 but not A/PR/8/34. This is probably because the two viruses differ in amino acid sequence at positions 408 and 411 within this part of the NP molecule. In contrast to the results observed with CD4-positive T cell epitopes, the major determinant recognized by CD8-positive T cells was only presented after live viral infection. The results in this study have important implications for vaccine design, inasmuch as they indicate that the same dominant CD4 T cell determinants on NP presented by vaccination with NP are also recognized by T cells from mice exposed to infectious virus.     

Immunoproteasome-deficient mice mount largely normal CD8+ T cell responses to lymphocytic choriomeningitis virus infection and DNA vaccination

During viral infection, constitutive proteasomes are largely replaced by immunoproteasomes, which display distinct cleavage specificities, resulting in different populations of potential CD8(+) T cell epitope peptides. Immunoproteasomes are believed to be important for the generation of many viral CD8(+) T cell epitopes and have been implicated in shaping the immunodominance hierarchies of CD8(+) T cell responses to influenza virus infection. However, it remains unclear whether these conclusions are generally applicable. In this study we investigated the CD8(+) T cell responses to lymphocytic choriomeningitis virus infection and DNA immunization in wild-type mice and in mice lacking the immunoproteasome subunits LMP2 or LMP7. Although the total number of virus-specific cells was lower in LMP2 knockout mice, consistent with their having lower numbers of naive cells before infection, the kinetics of virus clearance were similar in all three mouse strains, and LMP-deficient mice mounted strong primary and secondary lymphocytic choriomeningitis virus-specific CD8(+) T cell responses. Furthermore, the immunodominance hierarchy of the four investigated epitopes (nuclear protein 396 (NP(396)) > gp33 > gp276 > NP(205)) was well maintained. We observed a slight reduction in the NP(205)-specific response in LMP2-deficient mice, but this had no demonstrable biological consequence. DNA vaccination of LMP2- and LMP7-deficient mice induced CD8(+) T cell responses that were slightly lower than, although not significantly different from, those induced in wild-type mice. Taken together, our results challenge the notion that immunoproteasomes are generally needed for effective antiviral CD8(+) T cell responses and for the shaping of immunodominance hierarchies. We conclude that the immunoproteasome may affect T cell responses to only a limited number of viral epitopes, and we propose that its main biological function may lie elsewhere.     

A human multi-epitope recombinant vaccinia virus as a universal T cell vaccine candidate against influenza virus

There is a need to develop a universal vaccine against influenza virus infection to avoid developing new formulations of a seasonal vaccine each year. Many of the vaccine strategies for a universal vaccine target strain-conserved influenza virus proteins, such as the matrix, polymerase, and nucleoproteins, rather than the surface hemagglutinin and neuraminidase proteins. In addition, non-disease-causing viral vectors are a popular choice as a delivery system for the influenza virus antigens. As a proof-of-concept, we have designed a novel influenza virus immunogen based on the NP backbone containing human T cell epitopes for M1, NS1, NP, PB1 and PA proteins (referred as NPmix) as well as a construct containing the conserved regions of influenza virus neuraminidase (N-terminal) and hemagglutinin (C-terminal) (referred as NA-HA). DNA vectors and vaccinia virus recombinants expressing NPmix (WR-NP) or both NPmix plus NA-HA (WR-flu) in the cytosol were tested in a heterologous DNA-prime/vaccinia virus-boost vaccine regimen in mice. We observed an increase in the number of influenza virus-specific IFNγ-secreting splenocytes, composed of populations marked by CD4(+) and CD8(+) T cells producing IFNγ or TNFα. Upon challenge with influenza virus, the vaccinated mice exhibited decreased viral load in the lungs and a delay in mortality. These findings suggest that DNA prime/poxvirus boost with human multi-epitope recombinant influenza virus proteins is a valid approach for a general T-cell vaccine to protect against influenza virus infection.     

TIM-4, a receptor for phosphatidylserine, controls adaptive immunity by regulating the removal of antigen-specific T cells

Adaptive immunity is characterized by the expansion of an Ag-specific T cell population following Ag exposure. The precise mechanisms, however, that control the expansion and subsequent contraction in the number of Ag-specific T cells are not fully understood. We show that T cell/transmembrane, Ig, and mucin (TIM)-4, a receptor for phosphatidylserine, a marker of apoptotic cells, regulates adaptive immunity in part by mediating the removal of Ag-specific T cells during the contraction phase of the response. During Ag immunization or during infection with influenza A virus, blockade of TIM-4 on APCs increased the expansion of Ag-specific T cells, resulting in an increase in secondary immune responses. Conversely, overexpression of TIM-4 on APCs in transgenic mice reduced the number of Ag-specific T cells that remained after immunization, resulting in reduced secondary T cell responses. There was no change in the total number of cell divisions that T cells completed, no change in the per cell proliferative capacity of the remaining Ag-specific T cells, and no increase in the development of Ag-specific regulatory T cells in TIM-4 transgenic mice. Thus, TIM-4-expressing cells regulate adaptive immunity by mediating the removal of phosphatidylserine-expressing apoptotic, Ag-specific T cells, thereby controlling the number of Ag-specific T cells that remain after the clearance of Ag or infection.     

Identification and characterization of T helper epitopes in the nucleoprotein of influenza A virus

By using a series of overlapping synthetic peptides that cover more than 95% of the amino acid sequence of nucleoprotein (NP) of influenza A/NT/60/68 virus, five Th cell epitopes in B10.S (H-2s), BALB/c (H-2d), CBA (H-2k), and B6 (H-2b) mice have been identified. The specificity of Th cell recognition of epitopes is largely dependent on the H-2 haplotype of the responding mouse strain. However, two out of the five Th epitopes defined could be recognized by mice of more than one haplotype, implying that the primary sequence of protein antigens could also influence the selection of dominant T cell epitopes by the immune system. Immunization of B10.S mice with peptide 260-283 generated strong Th cell response against type A influenza viruses. In the other three strains of mice tested, priming with helper peptides induced a stronger antipeptide than antiviral T cell response. However, the low responsiveness to virus in these mice could be partially overcome by immunization with a mixture of several helper peptides. The Th epitopes are defined by the ability of the peptides to stimulate class II MHC restricted CD4+ T cells to proliferate and to produce IL-2 in vitro. When compared with the known epitopes on NP recognised by class I restricted CD8+ cytotoxic T cells, it appears that Th and cytotoxic T cell epitopes are nonoverlapping. The AMPHI and Motifs methods were employed to analyze the sequence of NP and predict the potential dominant sites in the molecule. The predictions are compared with the experimental data obtained and the implications discussed.     

Analysis of immune responses against nucleocapsid protein of the Hantaan virus elicited by virus infection or DNA vaccination

Even though neutralizing antibodies against the Hantaan virus (HTNV) has been proven to be critical against viral infections, the cellular immune responses to HTNV are also assumed to be important for viral clearance. In this report, we have examined the cellular and humoral immune responses against the HTNV nucleocapsid protein (NP) elicited by virus infection or DNA vaccination. To examine the cellular immune response against HTNV NP, we used H-2K(b) restricted T-cell epitopes of NP. The NP-specific CD8(+) T cell response was analyzed using a (51)Cr-release assay, intracellular cytokine staining assay, enzyme-linked immunospot assay and tetramer binding assay in C57BL/6 mice infected with HTNV. Using these methods, we found that HTNV infection elicited a strong NP-specific CD8(+) T cell response at eight days after infection. We also found that several different methods to check the NP-specific CD8(+) T cell response showed a very high correlation among analysis. In the case of DNA vaccination by plasmid encoding nucleocapsid gene, the NP-specific antibody response was elicited 2 approximately 4 weeks after immunization and maximized at 6 approximately 8 weeks. NP-specific CD8(+) T cell response reached its peak 3 weeks after immunization. In a challenge test with the recombinant vaccinia virus expressing NP (rVV-HTNV-N), the rVV-HTNV-N titers in DNA vaccinated mice were decreased about 100-fold compared to the negative control mice.     

Infection with seasonal influenza virus elicits CD4 T cells specific for genetically conserved epitopes that can be rapidly mobilized for protective immunity to pandemic H1N1 influenza virus

In recent years, influenza viruses with pandemic potential have been a major concern worldwide. One unresolved issue is how infection or vaccination with seasonal influenza virus strains influences the ability to mount a protective immune response to novel pandemic strains. In this study, we developed a mouse model of primary and secondary influenza infection by using a widely circulating seasonal H1N1 virus and the pandemic strain of H1N1 that emerged in Mexico in 2009, and we evaluated several key issues. First, using overlapping peptide libraries encompassing the entire translated sequences of 5 major influenza virus proteins, we assessed the specificity of CD4 T cell reactivity toward epitopes conserved among H1N1 viruses or unique to the seasonal or pandemic strain by enzyme-linked immunospot (ELISpot) assays. Our data show that CD4 T cells reactive to both virus-specific and genetically conserved epitopes are elicited, allowing separate tracking of these responses. Populations of cross-reactive CD4 T cells generated from seasonal influenza infection were found to expand earlier after secondary infection with the pandemic H1N1 virus than CD4 T cell populations specific for new epitopes. Coincident with this rapid CD4 T cell response was a potentiated neutralizing-antibody response to the pandemic strain and protection from the pathological effects of infection with the pandemic virus. This protection was not dependent on CD8 T cells. Together, our results indicate that exposure to seasonal vaccines and infection elicits CD4 T cells that promote the ability of the mammalian host to mount a protective immune response to pandemic strains of influenza virus.     

Multiple Distinct Forms of CD8+ T Cell Cross-Reactivity and Specificities Revealed after 2009 H1N1 Influenza A Virus Infection in Mice

Influenza primed mice are protected against lethal infection with H1N1 A/CA/04/E3/09 virus, and T depletion and serum transfer studies suggest a T-dependent mechanism. We therefore set out to investigate the quality of the cross-reactive T cell response to CA/E3/09 in mice primed with H3N2 influenza A/Hong Kong/X31 virus. Sequences of the immunodominant nucleoprotein (NP) NP366–374 and acid polymerase (PA) PA224–233 CD8 epitopes from X31 each differ from the CA/E3/09 virus by one amino acid: an M371V substitution at position 6 of the NP peptide, and an S224P substitution at position 1 of the PA peptide, raising questions about the role of these epitopes in protection. PA224–233 peptides from either virus could elicit IFN-γ spot forming cells from mice infected with X31, indicating cross-reactivity of these two peptides. However, no T cell responses to either PA224–233 peptide were detectable after primary CA/E3/09 infection, suggesting it is cryptic in this virus. In contrast, primary responses to the NP366 peptides were detectable after infection with either virus, but did not cross-react in vitro. Similarly, H2-D^b tetramers of each NP epitope stained CD8+ T cells from each respective virus infection, but did not obviously cross-react. Early after lethal CA/E3/09 challenge, X31 primed mice had enhanced IFN-γ responses toward both NP366 peptides, as well as recall responses to a set of subdominant NP and PA peptides not detectable after primary X31 infection alone. Furthermore, dual-tetramer staining revealed an expanded population of CD8 T cells reactive to both NP366 variant peptides also not seen after the priming infection alone. These observations demonstrate unusual CD8+ T cell cross-reactivity and specificity are elicited after primary and secondary CA/E3/09 influenza virus infections.     

Healthy human subjects have CD4+ T cells directed against H5N1 influenza virus

It is commonly perceived that the human immune system is naive to the newly emerged H5N1 virus. In contrast, most adults have been exposed to influenza A H1N1 and H3N2 viruses through vaccination or infection. Adults born before 1968 have likely been exposed to H2N2 viruses. We hypothesized that CD4(+) T cells generated in response to H1N1, H3N2, and H2N2 influenza A viruses also recognize H5N1 epitopes. Tetramer-guided epitope mapping and Ag-specific class II tetramers were used to identify H5N1-specific T cell epitopes and detect H5N1-specific T cell responses. Fifteen of 15 healthy subjects tested had robust CD4(+) T cell responses against matrix protein, nucleoprotein, and neuraminidase of the influenza A/Viet Nam/1203/2004 (H5N1) virus. These results are not surprising, because the matrix protein and nucleoprotein of influenza A viruses are conserved while the neuraminidase of the H5N1 virus is of the same subtype as that of the circulating H1N1 influenza strain. However, H5N1 hemagglutinin-reactive CD4(+) T cells were also detected in 14 of 14 subjects examined despite the fact that hemagglutinin is less conserved. Most were cross-reactive to H1, H2, or H3 hemagglutinin epitopes. H5N1-reactive T cells were also detected ex vivo, exhibited a memory phenotype, and were capable of secreting IFN-gamma, TNF-alpha, IL-5, and IL-13. These data demonstrate the presence of H5N1 cross-reactive T cells in healthy Caucasian subjects, implying that exposure to influenza A H1N1, H3N2, or H2N2 viruses through either vaccination or infection may provide partial immunity to the H5N1 virus.     

A dominant Th epitope in influenza nucleoprotein. Analysis of the fine specificity and functional repertoire of T cells recognizing a single determinant.

The sequence 260-283 of the nucleoprotein (NP) of influenza A virus is an epitope recognized by virus-immune lymph node cells from CBA (H-2k), B6 (H-2b), and B10.S (H-2s) mice. Further analysis shows that there are at least two Th epitopes within this sequence: the one close to the N-terminal (p260-273) is recognized by T cells from CBA and B6 mice while that close to the carboxyl-terminal (p270-283) is a dominant Th determinant in B10.S mice. The fine specificity of the recognition of this epitope by NP-specific T cell clones is also studied. When B10.S mice were infected intranasally or i.v. with live influenza virus, or immunized by different ways with various Ag preparations, P270-283 persistently emerged as a dominant T cell epitope. Immunization of B10.S mice with peptide p270-283 induces T cells with different in vivo functions including class II-restricted cytotoxicity, cognate help for Ag-specific antibody synthesis and delayed type hypersensitivity. This may have important implications for the understanding of the differentiation and classification of subsets of CD4+ T cells. The corresponding sequence of the NP of an equine influenza virus, A/Eq/Prague/56, which has a substitution (leucine to proline) at position 283, was not recognized by the lymph node cells from mice primed with either A/Okuda or A/Eq/Prague. However, the peptide, p270-283(E), representing this sequence induced T cell responses to both human and equine viruses. The data are discussed with respect to the development of viral vaccines.     

Enumeration of antigen-presenting cells in mice infected with Sendai virus

Substantial progress has been made in understanding Ag presentation to T cells; however, relatively little is known about the location and frequency of cells presenting viral Ags during a viral infection. Here, we took advantage of a highly sensitive system using lacZ-inducible T cell hybridomas to enumerate APCs during the course of respiratory Sendai virus infection in mice. Using lacZ-inducible T cell hybridomas specific for the immunodominant hemagglutinin-neuraminidase HN421-436/I-Ab and nucleoprotein NP324-332/Kb epitopes, we detected APCs in draining mediastinal lymph nodes (MLNs), in cervical lymph nodes, and also in the spleen. HN421-436/I-Ab- and NP324-332/Kb-presenting cells were readily detectable between days 3 and 9 postinfection, with more APCs present in the MLN than in the cervical lymph nodes. Interestingly, no infectious virus was detected in lymphoid tissue beyond day 6, suggesting that a depot of noninfectious viral Ag survives, in some form, for 2-3 days after viral clearance. Fractionation of the MLN demonstrated that APC frequency was enriched in dendritic cells and macrophages but depleted in the B cell population, suggesting that B cells do not form a large population of APCs during the primary response to this virus.     

Hidden epitopes emerge in secondary influenza virus-specific CD8+ T cell responses

Influenza A virus-specific CD8+ T cell responses in H2(b) mice are characterized by reproducible hierarchies. Compensation by the D(b)PB1-F2(62) epitope is apparent following infection with a variant H3N2 virus engineered to disrupt the prominent D(b)NP(366) and D(b)PA(224) epitopes (a double knockout or DKO). Analysis with a "triple" knockout (TKO) virus, which also compromises D(b)PB1-F2(62), did not reveal further compensation to the known residual, minor, and predicted epitopes. However, infection with this deletion mutant apparently switched protective immunity to an alternative Ab-mediated pathway. As expected, TKO virus clearance was significantly delayed in Ab-deficient MHC class II(-/-) and Ig(-/-) mice, which were much more susceptible following primary, intranasal infection with the TKO, but not DKO, virus. CD8+ T cell compensation was detected in DKO, but not TKO, infection of Ig-deficient mice, suggestive of cooperation among CD8+ T cell responses. However, after priming with a TKO H1N1 mutant, MHC II(-/-) mice survived secondary intranasal exposure to the comparable H3N2 TKO virus. Such prime/challenge experiments with the DKO and TKO viruses allowed the emergence of two previously unknown epitopes. The contrast between the absence of compensatory effect following primary exposure and the substantial clonal expansion after secondary challenge suggests that the key factor limiting the visibility of these "hidden" epitopes may be very low naive T cell precursor frequencies. Overall, these findings suggest that vaccine approaches using virus vectors to deliver an Ag may be optimized by disrupting key peptides in the normal CD8+ T cell response associated with common HLA types.     

Synthetic Long Peptide Influenza Vaccine Containing Conserved T and B Cell Epitopes Reduces Viral Load in Lungs of Mice and Ferrets

Currently licensed influenza vaccines mainly induce antibodies against highly variable epitopes. Due to antigenic drift, protection is subtype or strain-specific and regular vaccine updates are required. In case of antigenic shifts, which have caused several pandemics in the past, completely new vaccines need to be developed. We set out to develop a vaccine that provides protection against a broad range of influenza viruses. Therefore, highly conserved parts of the influenza A virus (IAV) were selected of which we constructed antibody and T cell inducing peptide-based vaccines. The B epitope vaccine consists of the highly conserved HA2 fusion peptide and M2e peptide coupled to a CD4 helper epitope. The T epitope vaccine comprises 25 overlapping synthetic long peptides of 26-34 amino acids, thereby avoiding restriction for a certain MHC haplotype. These peptides are derived from nucleoprotein (NP), polymerase basic protein 1 (PB1) and matrix protein 1 (M1). C57BL/6 mice, BALB/c mice, and ferrets were vaccinated with the B epitopes, 25 SLP or a combination of both. Vaccine-specific antibodies were detected in sera of mice and ferrets and vaccine-specific cellular responses were measured in mice. Following challenge, both mice and ferrets showed a reduction of virus titers in the lungs in response to vaccination. Summarizing, a peptide-based vaccine directed against conserved parts of influenza virus containing B and T cell epitopes shows promising results for further development. Such a vaccine may reduce disease burden and virus transmission during pandemic outbreaks.     

Strong memory CD8+ T cell responses against immunodominant and three new subdominant HLA-B27-restricted influenza A CTL epitopes following secondary infection of HLA-B27 transgenic mice

We previously showed that the known HLA-B27-restricted influenza A epitope identified from human studies, NP.383-391, was recognized by CTLs following influenza A infection of transgenic (Tg) HLA-B27/H2 class I-deficient (H2 DKO) mice. Here, we examined the kinetics of the primary NP.383-391-specific response in Tg HLA-B27/H2 DKO mice at the site of respiratory infection, along with the profile of additional influenza A epitopes recognized. While the temporal kinetics of the Tg HLA-B27/NP.383-391-specific CD8+ T cell response paralleled the H2-D(b)/NP.366-374-specific response of non-Tg H2b mice, the magnitude was less. Using epitope prediction programs, we identified three novel B27-restricted influenza A epitopes, PB2.702-710, PB1.571-579, and PB2.368-376, recognized during both the primary and secondary response to infection. Although the secondary NP.383-391-specific response was dominant, PB1.571-579 and PB2.368-376 stimulated stronger proliferative expansion in memory T cells. Our results indicate a broader B27/influenza A CTL repertoire than previously known. Together with results for other HLA class I alleles, this information will become important in improving vaccine strategies for influenza A and other human pathogens.     

The context of epitope presentation can influence functional quality of recalled influenza A virus-specific memory CD8+ T cells

Lipopeptide constructs offer a novel strategy for eliciting effective cellular and humoral immunity by directly targeting the vaccine Ag to dendritic cells. Importantly, it is not known how closely immunity generated after lipopeptide vaccination mimics that generated after natural infection. We have used a novel lipopeptide vaccine strategy to analyze both the quantity and quality of CD8(+) T cell immunity to an influenza A virus epitope derived from the acidic polymerase protein (PA(224)) in B6 mice. Vaccination with the PA(224) lipopeptide resulted in accelerated viral clearance after subsequent influenza virus infection. The lipopeptide was also effective at recalling secondary D(b)PA(224) responses in the lung. Lipopeptide recalled D(b)PA(224)-specific CTL produced lower levels of IFN-gamma and TNF-alpha, but produced similar levels of IL-2 when compared with D(b)PA(224)-specific CTL recalled after virus infection. Furthermore, lipopeptide- and virus-recalled CTL demonstrated similar TCR avidity. Interestingly, lipopeptide administration resulted in expansion of D(b)PA(224)-specific CTL using a normally subdominant TCRBV gene segment. Overall, these results demonstrate that protective CTL responses elicited by lipopeptide vaccines can be correlated with TCR avidity, IL-2 production, and broad TCR repertoire diversity. Furthermore, factors that impact the quality of immunity are discussed. These factors are important considerations when evaluating the efficacy of novel vaccine strategies that target dendritic cells for eliciting cellular immunity.     

Antigen-specific CD8(+) T cells persist in the upper respiratory tract following influenza virus infection.

Because little is known about lymphocyte responses in the nasal mucosa, lymphocyte accumulation in the nasal mucosa, nasal-associated lymphoid tissue (NALT), and cervical lymph nodes (CLN) were determined after primary and heterosubtypic intranasal influenza challenge of mice. T cell accumulation peaked in the nasal mucosa on day 7, but peaked slightly earlier in the CLN (day 5) and later (day 10) in the NALT. Tetrameric staining of nasal mucosal cells revealed a peak accumulation of CD8 T cells specific for either the H-2D(b) influenza nucleoprotein epitope 366-374 (D(b)NP(366)) or the H-2D(b) polymerase 2 protein epitope 224-233 (D(b)PA(224)) at 7 days. By day 13, D(b)PA(224)-specific CD8 T cells were undetectable in the mucosa, whereas D(b)NP(366)-specific CD8 T cells persisted for at least 35 days in the mucosa and spleen. After heterosubtypic virus challenge, the accumulation of CD8 T cells in the nasal mucosa was quicker, more intense, and predominantly D(b)NP(366) specific relative to the primary inoculation. The kinetics and specificity of the CD8 T cell response were similar to those in the CLN, but the responses in the NALT and spleen were again slower and more protracted. These results indicate that similar to what was reported in the lung, D(b)NP(366)-specific CD8 T cells persist in the nasal mucosa after primary influenza infection and predominate in an intensified nasal mucosal response to heterosubtypic challenge. In addition, differences in the kinetics of the CD8 T cell responses in the CLN, NALT, and spleen suggest different roles of these lymphoid tissues in the mucosal response.