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The current framework for testing and regulating vaccines was established before the realization that vaccines, in addition to their effect against the vaccine-specific disease, may also have “non-specific effects” affecting the risk of unrelated diseases. Accumulating evidence from epidemiological studies shows that vaccines in some situations can affect all-cause mortality and morbidity in ways that are not explained by the prevention of the vaccine-targeted disease. Live attenuated vaccines have sometimes been associated with decreases in mortality and morbidity that are greater than anticipated. In contrast, some non-live vaccines have in certain contexts been associated with increases in all-cause mortality and morbidity. The non-specific effects are often greater for female than male individuals. Immunological studies have provided several mechanisms that explain how vaccines might modulate the immune response to unrelated pathogens, such as through trained innate immunity, emergency granulopoiesis, and heterologous T-cell immunity. These insights suggest that the framework for the testing, approving, and regulating vaccines needs to be updated to accommodate non-specific effects. Currently, non-specific effects are not routinely captured in phase I–III clinical trials or in the post-licensure safety surveillance. For instance, an infection with Streptococcus pneumoniae occurring months after a diphtheria-tetanus-pertussis vaccination would not be considered an effect of the vaccination, although evidence indicates it might well be for female individuals. Here, as a starting point for discussion, we propose a new framework that considers the non-specific effects of vaccines in both phase III trials and post-licensure. The current framework for testing and regulating vaccines was established before the realization that vaccines, in addition to their effect against the vaccine-specific disease, may also have "non-specific effects" affecting the risk of unrelated diseases. Accumulating evidence from epidemiological studies shows that vaccines in some situations can affect all-cause mortality and morbidity in ways that are not explained by the prevention of the vaccine-targeted disease. Live attenuated vaccines have sometimes been associated with decreases in mortality and morbidity that are greater than anticipated. In contrast, some non-live vaccines have in certain contexts been associated with increases in all-cause mortality and morbidity. The non-specific effects are often greater for female than male individuals. Immunological studies have provided several mechanisms that explain how vaccines might modulate the immune response to unrelated pathogens, such as through trained innate immunity, emergency granulopoiesis, and heterologous T-cell immunity. These insights suggest that the framework for the testing, approving, and regulating vaccines needs to be updated to accommodate non-specific effects. Currently, non-specific effects are not routinely captured in phase I-III clinical trials or in the post-licensure safety surveillance. For instance, an infection with Streptococcus pneumoniae occurring months after a diphtheria-tetanus-pertussis vaccination would not be considered an effect of the vaccination, although evidence indicates it might well be for female individuals. Here, as a starting point for discussion, we propose a new framework that considers the non-specific effects of vaccines in both phase III trials and post-licensure.

For unknown reasons 5-10% of middle-aged adults (>35 years) remain EBV-sero-negative when the virus infects the vast majority of people, and is actively shed at high titers during chronic infection. Here we show that EBV-sero-negative (EBV-SN) HLA-A2+ middle-aged adults possess a unique IAV-M158-66(GIL) memory CD8 T cell response that cross-reacts with EBV lytic epitopes. The five tested HLA-A2+ EBV-SN middle-aged adults had a significantly increased IAV-M158-66(GIL) tetramer+ CD8 frequency compared to adult EBV sero-positive donors. Upon exposure to EBV antigens in vitro both IAV- M1(GIL) /EBV-BMLF1280-288(GLC) and IAV- M1(GIL) /EBV-BRLF1109-117(YVL), functionally cross-reactive CD8+ responses could be detected in the peripheral blood of middle-aged EBV-SN donors, while only weakly IAV-(M1)/EBV-(YVL) cross-reactive responses were detected in 4 out of 6 teenage EBV-SN donors. These IAV-M1-GIL-specific CD8 T cells in middle-aged EBV-SN adults expanded dramatically to EBV lytic antigens and produced cytokines at high functional avidity. They lysed EBV-infected targets. Additionally, these cross-reactive cells had an oligo-clonal T cell receptor repertoire different than EBV sero-positive donors. Taken together these data suggest that an altered cross-reactive T cell repertoire could mediate protective immunity against viral infection. Our results imply that sero-negative adults might have the ability to resist viral infection via heterologous immunity.

Activation of crossreactive memory CD8 T cells leads to heterologous protective immunity. Here we question whether early activation of crossreactive memory CD8 T cells alters the activation of new naïve CD8 responses to the second virus. Pichinde virus (PICV) and lymphocytic choriomeningitis virus (LCMV) contain a crossreactive usually subdominant epitope, NP205, which differs in 6 of 8 amino acids. During PICV infection of LCMV-immune mice the NP205 memory response become immunodominant and mediates protective immunity. PICV is cleared early and does not persist. The usually dominant non-crossreactive new naïve PICV NP38 CD8 T cell response appears to be subdominant day 7 post infection. However, surprisingly over the next 100 days post infection the NP38 response continues to gradually expand and then stabilizes. These NP38-specific CD8 T cells appear to arrest in an intermediate effector T cell phenotype (KLRG1low, CD62Llow IL-7Rhi), with altered functionality (decreased TNFα production). They appear to have altered expression of apoptotic genes on gene array. This NP38 inflationary response did not occur when mice were immunized with an LCMV mutant that did not present the crossreactive NP205 epitope. Our data shows that the strong crossreactive response kills DC’s expressing NP205 early (day 2-3) in PICV infection. This may result in an early termination of antigen-presentation leading to inadequate activation of the naïve NP38 response and inflation.

IAV is a common human respiratory pathogen with a strong association with heterotypic and heterologous immunity. IAV-immune mice challenged with LCMV develop detrimental effects with both increased virus titers and immunopathology. Crossreactive CD8 T cell responses between these two viruses mediate the enhanced lung pathology. Here, we questioned whether crossreactive CD4 memory cells in IAV-immune mice could play a role in increasing viral titers during the subsequent LCMV infection. Preferential depletion of memory CD4 but not CD8 cells in IAV-immune mice prevented the increase in LCMV titers. In IAV+LCMV mice there was an early increase in CD4 cells with a shift in the CD4/CD8 ratio. Luminex cytokine assays suggested that there was an increase in Th2 cytokines, IL-4 and IL-5, in the spleen of IAV+LCMV mice, which was IAV CD4 memory cell dependent, as IL-4 decreased in the CD4 depleted mice. Depleting IL-4 or IL-5 returned LCMV titers to naïve levels while adding rIL-4 and rIL-5 together to naïve mice increased virus titers. This suggests that the crossreactive heterologous response was more Th2-polarized and that since memory cytokines are produced early they may shift the normally strong Th1-type response to LCMV towards a more Th2-type response, inhibiting appropriate CD8 activation and impeding viral clearance. This shift to stronger Th2-type response would contribute to detrimental effects as LCMV requires a strong CD8 response and perforin to clear.

During EBV-associated infectious mononucleosis (IM) IAV-specific crossreactive memory T cells are activated and play a role in disease severity. In HLA-A2+ IM patients, influenza M158 (IAV-M1)-specific CD8 memory T cell responses crossreacted with two different EBV lytic epitopes, BMLF1280 (17/29) and BRLF1109 (19/20). Disease severity of IM directly correlated with significantly increased frequencies of crossreactive IAV-M1/EBV-BMLF1, IAV-M1, and EBV-BMLF1 specific CD8 cells, and with mean viral load over the first 5 weeks of infection. Disease severity did not correlate with BRLF1 or M1/BRLF1 crossreactive responses. When severity of IM was scored and patients were assigned to either mild or severe groups, disease severity correlated with specific TCR Vb usage in IAV-M1 population suggesting that TcR selection is driving disease outcome. Consistent with crossreactive responses driving increased immunopathology was the observation that tetramer-sorted crossreactive responses had completely altered immune response signatures than cognate responses by gene array. These results suggest that T cell crossreactivity impacts T cell selection and function and ultimately disease outcome. Insights on these issues are important for the intelligent design of vaccines and to develop therapeutic interventions for virally induced disease (NIHAI49320).

The competence of T cell responses predominantly depends on how efficient T cell receptors (TCRs) are at recognizing antigenic epitopes. We show here that during acute infectious mononucleosis (IM) there was an expansion of IAV-M1-specific memory cells that are crossreactive with both EBV-BMLF1 and EBV-BRLF1 double-tetramer+ cells directly ex-vivo in HLA-A2+ patients. These crossreactive populations expanded dramatically in culture. For instance, there were multiple crossreactive populations in the IAV-M1 stimulated line. We systematically examined the characteristics of these crossreactive populations and compared them to the cognate IAV-M1, EBV-BMLF and -BRLF1 populations using TCR deep sequencing and gene array analysis of tetramer sorted cells. Surprisingly, both crossreactive and non-crossreactive populations were highly polyclonal with 1500-3500 different VA and VB clonotypes. The dominant clonotypes differed between the different populations. However, there were overlaps in either VB or VA usage consistent with their ability to bind both ligands. In one patient the M1-specific response used was an unusual dominant VB and was identical with the BMLF1-specific VB (VB2J2.7) but they differed in VA with M1 using VA13J50.1 and BMLF1 VA 12.1J12.1. Immune response signatures from gene array differed between the crossreactive and non-crossreactive populations consistent with the concept that crossreactive TCR selection may play a role in disease outcome. (NIHAI49320).

Crossreactive CD8+T cells that recognize both influenza A (IAV) epitope M158, and Epstein Barr virus (EBV) epitope BRLF1109, are frequently found in HLA-A*02 populations particularly during acute infection. However, whether this crossreactivity is mediated by a single crossreactive T cell receptor (TCR) is unclear. To address this, M1- and BRLF1-MHCI oligomer positive CD8+T cells from peripheral blood of an acute IAV-infected patient who was EBV seropositive were single cell sorted and the TCR genes were sequenced and cloned. Thirty-three of 96 single cells yielded one TCRα and one TCRβ gene with 13 unique reproducible TCRα/β pairs. These 13 pairs of TCR genes were characterized in TCR deficient Jurkat cells expressing CD8α (J76) for antigen recognition and TCR signaling. In each case the transfected J76 cells bound strongly to M1-MHCI tetramer and weakly to BRLF1-MHCI tetramer consistent with the original staining in the peripheral blood. These crossreactive TCR-transfected J76 cells responded functionally to both peptides as they upregulated CD69 in response to BRLF1 as well as M1 peptide stimulation with four orders of magnitude difference. Interestingly, one TCR similar to JM22 which is a public TCR recognizing M1 epitope, was also crossreactive to BRLF1, which implies that many HLA-A*02 populations may be crossreactive to EBV. Our data clearly shows that crossreactivity between M1 and BRLF1 epitopes can be mediated by a single TCR and may be ubiquitous.

We previously showed that crossreactivity is common between IAV and EBV in HLA-A2+ patients during infectious mononucleosis. IAV-M1 specific CD8 T cells, along with expanded populations of IAV-M1/EBV-BRLF-1 and IAV-M1/EBV-BMLF-1 double-tetramer+ cells were detected directly ex-vivo in 5 HLA-A2+ patients. Altered IAV-M1, EBV-BRLF1 and -BMLF1 TCR repertoires were observed over the course of infection and in comparison to healthy donors. After culture, cells were sorted and analyzed by gene array in order to assess global changes in immune responses following different stimulations, either cognate or crossreactive, in different patient populations. M1- and BRLF1-specific cells had similar immune-response gene signatures, but the BMLF1-specific CD8 cells were more similar to the two-crossreactive populations. Crossreactive M1/BRLF1 cells from the BRLF1 line were different in their activation status than the BRLF1-specific cells, consistent with BRLF1 ligand stimulation of different gene activation profiles in these two populations. These results suggest that during symptomatic IAV infection there is an expansion of EBV/IAV crossreactive memory CD8 T cell responses. Ongoing studies are investigating whether EBV-IAV cross-reactive CD8+ T cells may contribute to immunopathology during acute IAV infection.

The competence of T cell responses predominantly depends on how efficient T cell receptors (TCRs) are at recognizing antigenic epitopes. We show here that during acute severely symptomatic IAV infection there was an expansion of IAV-M1/EBV-BRLF1 and IAV-M1/EBV-BMLF1 double-tetramer+ cells directly ex-vivo in 5 HLA-A2+ patients. We questioned whether this expansion specific to these two different crossreactive responses would lead to alterations in the TCR repertoire of the IAV-M1-58, EBV-BRLF1-109 and BMLF1-280 from before, during and following acute IAV infection. Using staining with Vb MAb we found that T cell responses generated to these epitopes became surprisingly more polyclonal, with the sharing of Vb between M1, BMLF1 and BRLF1 populations which is not seen in healthy donors and which decreased 2 months later consistent with crossreactive expansion. Furthermore, by using single-cell analysis of TCRα and TCRβ repertoire of tetramer sorted IAV-M1 cells we showed dramatic changes in specific clonotype usage and in JA and JB family usage during acute IAV infection compared to before infection. In summary, these changes in TCR repertoire during acute symptomatic IAV infection suggest that during severe infection there is a preferential expansion of highly diverse crossreactive responses between IAV and the persistent virus, EBV, which leads to permanent changes in TCR repertoires to both of these two viruses.

Newborns are more susceptible to infections due to their lack of immunological memory and under-developed immune systems. Passive maternal immunity protects neonates until their immune systems have matured. We questioned if a noncytolytic virus that produces strong T cell responses in adults would also induce a response in neonates. Neonates were infected with LCMV at 5e4, 500, 50, 5 and 0.5 PFU. Surprisingly, at all doses the majority of pups succumbed to infection during the peak of the T cell response. Death was caused by T cell-dependent pathology and not viral load as 100% of T cell deficient pups survived with minimal lung and liver pathology. However, passive immunity from LCMV-immune mothers protected 100% of pups from death by helping control viral load early in infection. In adult mice high antigenic stimulation can cause over-activation of T cells leading to clonal exhaustion, where cells undergo a stepwise loss of function. Clonal exhaustion allows for survival from some high dose viral infections and results in persistent infection. We found that neonates without passive immunity that survived to day 14 did not show signs of T cell clonal exhaustion. Furthermore, surviving pups were not persistently infected, clearing virus by week 3. Neonates are commonly thought to have less functional immature immune systems, but these results show that neonates are capable of producing strong T cell responses that contribute to increased mortality.

Alterations in memory CD8 T cell responses may contribute to the high morbidity and mortality caused by seasonal influenza A virus (IAV) infections in older individuals. We questioned whether memory CD8 responses changed overtime with increasing age to this non-persistent virus, to which recurrent exposure with new strains is common. Here, for the first time we show that with age the HLA-A2-restricted IAV M158-66-specific Vα T cell repertoire was significantly narrowed leading to oligoclonal expansions including the usage of a single identical VA12 clonotype in all 8 older donors. The VA repertoire of older individuals also had longer CDR3 regions with increased usage of alanine/glycine runs which may enhance TcR promiscuity. Collectively these results suggest that CD8 memory responses in humans to non-persistent viruses like IAV are dynamic, and with aging become narrower with preferential retention of T cell repertoires with features of enhanced promiscuity.

The neonatal immune system is immature, lacks memory and is undergoing tolerization. This is also the age when children receive multiple vaccines. Previously, we have shown that sequential infection with unrelated pathogens can alter disease outcome. This is defined as heterologous immunity and is mediated by crossreactive T cells. In adult mice previous exposure to LCMV protects mice from lethal vaccinia virus (VV) infection. Crossreactive LCMV-specific memory cells recognize VV epitopes, are reactivated and expand during VV infection. This causes faster viral clearance and a skewing of the LCMV-specific immunodominance hierarchy. However, some LCMV-immune mice also develop acute fatty necrosis (AFN) of the abdominal fat pads during a VV infection. We questioned if mice immunized as neonates would have protective heterologous immunity during VV challenge. Mice immunized as neonates had comparable reduction in VV load and induction of AFN indicating that heterologous immunity is established during viral infections early in life. Interestingly, the LCMV-specific memory populations that expanded in mice immunized as neonates differed from that of mice immunized as adults. In adult mice 50% of the mice have an expansion of LCMV-NP205-specific CD8 T cells while the majority of neonates expanded the LCMV-GP34-specific CD8 T cell pool. This alteration in dominant crossreactivities may be due to the limited T cell receptor repertoire of neonatal mice.

During EBV-associated IM influenza A-specific crossreactive memory T cells are activated and play a role in disease severity. In HLA-A2+ IM patients, influenza M158 (IAV-M1)-specific CD8 memory T cell responses cross-reacted with two different EBV lytic epitopes, BMLF-1280 (17/29) and BRLF-1190 (19/20). Furthermore, 11/22 IM patients demonstrated some intra-viral cross-reactivity between EBV-BRLF1 and -BMLF1 responses. Disease severity of IM did not correlate with viral load, but instead directly correlated with significantly increased frequencies of crossreactive IAV-M1/EBV-BMLF-1-specific CD8 cells, as well as IAV-M1, EBV-BMLF-1 specific CD8 cells, but not BRLF1 responses. When severity of IM was scored and patients were assigned to either mild or severe groups, disease severity correlated with specific TCR Vb usage in all three tetramer-positive populations suggesting that TcR selection is driving disease outcome. Consistent with IAV-M1 and EBV-BMLF1 responses driving increased immunopathology was the observation that patients with severe disease had significantly more IAV-M1 and EBV-BMLF1 cells producing IFNg/MIP1-b in response to antigen as compared to patients with mild disease. These results suggest that T cell crossreactivity can impact T cell selection and function and ultimately disease outcome. Insights on these issues are important for the intelligent design of vaccines and to develop effect therapeutic interventions for virally induced disease.

Co-infection with multiple pathogens can result in increased pathology and difficulties with diagnosis and treatment. Moreover, interference between co-administered vaccines has resulted in lower antibody production. However, the effect of co-infections on T cell responses remains poorly understood. We have shown that sequential infection with unrelated viruses can alter T cell responses and disease outcome. This is defined as heterologous immunity, which is mediated by crossreactive T cells. We questioned if co-infection with two viruses containing a crossreactive epitope would alter the immune response and disease outcome upon rechallenge. Compared to mice infected with a single virus, rechallenged of mice co-infected with lymphocytic choriomenigitis (LCMV) and Pichinde viruses (PV) had a loss of immunoprotection and enhanced immunopathology. Each viral rechallenge identified a different potential mechanism to explain these changes. First, co-infection resulted in fewer LCMV-specific effector-like memory CD8 T cells leading to decreased protection and enhanced liver pathology. Secondly, co-infection caused some mice to have inappropriately enhanced crossreactive responses, which contributed to increased immunopathology. As combined vaccines or simultaneous immunizations are also co-infections, our data would suggest that T cell responses should be examined in the context of vaccine development.

The gamma-herpes Epstein-Barr virus (EBV) infects ~90% of individuals globally, establishing a life-long infection. The clinical presentation of EBV infection can range from asymptomatic to severe, occasionally fatal, acute infectious mononucleosis (IM). There is also a strong causal relationship between EBV infection and common malignancies, including carcinoma’s, and Burkitt’s lymphoma. However it is unknown why 5-10% of adults remain EBV-sero-negative (EBV-SN), despite the fact the virus infects the vast majority of the population and is actively shed at high titers even during chronic infection. Here, we show that EBV-SN HLA-A2+ adults possess cross-reactive IAV-GIL/EBV-GLC memory CD8 T-cells that show highly unique properties. These IAV-GIL crossreactive cells preferentially expand, and produce cytokines to, EBV antigens with high avidity. Additionally they are capable of lysing EBV-infected targets and show the potential to enter the mucosal epithelial tissue where infection is thought to initiate. They also possess a T-cell receptor (TCR) repertoire that differs by both organization and CDR3 usage from that in EBV-seropositive (EBV-SP) donors. Our results imply that heterologous immunity may protect EBV-SN adults against the establishment of productive EBV infection, and thus be the first demonstration of heterologous immunity between unrelated pathogens in sero-negative human subjects.

Alterations in memory CD8 T cell responses may contribute to the high morbidity and mortality caused by seasonal influenza A virus (IAV) infections in older individuals. We questioned whether memory CD8 responses changed overtime with increasing age. Here, for the first time we show that the HLA-A2-restricted IAV M158-66-specific Vα T cell repertoire was significantly narrowed leading to oligoclonal expansions including the usage of a single identical VA12 clonotype. This was the case for all 8 older donors. The VA repertoire of older individuals also had longer CDR3 regions with increased usage of alanine/glycine runs which may enhance TCR promiscuity. Collectively these results suggest that CD8 memory responses in humans to non-persistent viruses like IAV are dynamic, and become narrower with preferential retention of T cell repertoires with features of enhanced promiscuity with age.

Natural killer (NK) cells directly control virus replication during some infections and are postulated to indirectly regulate antiviral immunity during other infections through modulation of antiviral T cell responses, but the consequences and mechanisms of this regulation remain largely unexplored. Using the relatively NK cell-resistant lymphocytic choriomeningitis virus (LCMV), we identified a crucial role for NK cells in repression of antiviral T cell responses to a degree that alters the balance between virus clearance and immune pathology. Depletion of NK cells prior to infection of mice with a LCMV clone 13 dose (2 x 105 PFU) normally associated with fatal T cell-mediated immunopathology completely prevented virus-associated morbidity and promoted rapid viral clearance. In contrast, mice depleted of NK cells succumbed to a higher viral dose (2 x 106 PFU) normally associated with non-lethal persistent infection and clonal exhaustion of T cells. During the first three days of infection, NK cells eliminated activated CD4 T cells in a perforin-dependent manner. This early NK cell-mediated decrease in CD4 T cells resulted in lower numbers and diminished cytokine-producing capacity of LCMV-specific CD8 T cells. Thus, NK cells can dramatically alter viral clearance and infection-associated immunopathology by reducing the ability of CD4 T cells to support antiviral CD8 T cell responses in the context of high viral loads and disseminated infection.

T cell cross-reactivity describes the phenomenon whereby a single T cell can recognize two or more different peptide antigens presented in complex withMHCproteins. Cross-reactive T cells have previously been characterized at the population level by cytokine secretion and MHC tetramer staining assays, but single-cell analysis is difficult or impossible using these methods. In this study, we describe development of a novel peptide-MHC heterodimer specific for cross-reactive T cells. MHC-peptide monomers were independently conjugated to hydrazide or aldehyde-containing cross-linkers using thiol-maleimide coupling at cysteine residues introduced into recombinant MHC heavy chain proteins. Hydrazone formation provided bi-specific MHC heterodimers carrying two different peptides. Using this approach we prepared heterodimers of the murine class I MHC protein H-2Kb carrying peptides from lymphocytic choriomeningitis virus and vaccinia virus, and used these to identify crossreactive CD8[|#1#|] T cells recognizing both lymphocytic choriomeningitis virus and vaccinia virus antigens. A similar strategy could be used to develop reagents to analyze cross-reactive T cell responses in humans.

During EBV-associated infectious mononucleosis (IM) influenza A-specific crossreactive memory T cells are highly activated and play a role in disease severity. In HLA-A2+ IM patients, influenza M158 (IAV-M1)-specific CD8 memory T cell responses cross-reacted with two different EBV lytic epitopes, BRLF-1(190) (19/20) and BMLF-1(280) (17/29). Furthermore, 11/22 IM patients demonstrated some intra-viral cross-reactivity between EBV-BRLF-1 and -BMLF-1 responses. Disease severity of IM did not correlate with viral load, but instead directly correlated with the percentage of IAV-M1, EBV-BMLF-1 and crossreactive IAV-M1/EBV-BMLF-1-specific CD8 T cells. This was associated with altered TCR Vbeta usage. In all IM patients cross-reactive T cell responses were observed but different patterns of cross-reactivity were seen in each individual. A number of unique features of the TCR of the highly cross-reactive BRLF-1 epitope were noted. BRLF-1 T cell responses were of very high avidity. There was tremendous variation in the Vbeta usage between different individuals and even in the same individual over time. Cross-reactive BRLF-1 responses only became apparent at higher antigen doses in vitro, such as might occur during different phases of infectious mononucleosis.

Cross-reactive memory T cells specific to previously encountered influenza A contribute to the characterisitic lymphoproliferation of EBV-associated infectious mononucleosis (IM). In HLA-A2+ IM patients, influenza M158 (IAV-M1)-specific CD8 memory T cell responses cross-reacted with two different EBV lytic epitopes, BRLF-1190 (10/10) and BMLF-1280 (14/26). Disease severity of IM did not correlate with viral load. Interestingly, instead disease severity directly correlated with the percentage of Flu-M1-specific CD8 T cells but not with either EBV-BMLF-1, or -BRLF-1-specific responses. In addition, 8/19 IM patients demonstrated some intra-viral cross-reactivity between EBV-BRLF-1 and -BMLF-1 responses. These cross-reactive T cell responses were observed in all IM patients but different patterns of cross-reactivity occurred in each individual concerning the specific epitopes involved and their time of appearance after infection. A number of unique features of the TCR of the highly cross-reactive BRLF-1 epitope were noted. BRLF-1 T cell responses were of very high avidity. There was tremendous variation in the Vbeta/Valpha usage between different individuals, even in the same individual over time and this could be further modulated by peptide concentration in vitro. Cross-reactive BRLF-1 responses only became apparent at high antigen doses such as might occur during different phases of IM.

Heterologous immunity may help explain why any respiratory infection can cause a wide range of disease from asymptomatic to extremely severe, even resulting in death. Prior immunity to influenza A led to significantly increased viral load and lung pathology upon LCMV infection, the severity of which varied between genetically identical mice, but did not correlate with viral load. Flu-specific memory CD8 T cells mediated this increased pathology as evidenced by CD8 depletion studies and the observation that disease severity directly correlated with the expansion of two cross-reactive Flu-epitope specific memory CD8 T cell responses during LCMV infection. H2-Kb-restricted IAV-PB1703 and H2-Db-restricted IAV-PA224 responses cross-reacted with LCMV-GP34, and -GP276, respectively. Functionally inactivating these memory populations by peptide tolerization or blocking IFNγ activity greatly decreased lung pathology demonstrating novel therapeutic interventions. Flu-specific memory CD4 T cells mediated the enhanced viral load during LCMV infection as demonstrated by CD4 depletion studies and depletion of the early enhanced Th2 cytokine IL-4. These findings have important implications for understanding and potentially controlling variability in disease outcome during respiratory infections.

SUMMARY Administration of FSH to hypophysectomized male rats has been shown to increase the capacity of the testes to secrete testosterone in response to LH. Studies were done to assess the effect of increased endogenous FSH on the response of the testis to LH. At 17 and 24 days of age, animals hemiorchidectomized at 10 days of age had significantly higher serum FSH levels and testicular weights than sham-operated rats. To study the testicular response to LH in vivo, hemiorchidectomized animals were sham-operated and the sham-operated animals were hemiorchidectomized at 17 or 24 days of age. Luteinizing hormone (30 μg/100 g body wt) or saline was administered immediately after the operation and the animals were autopsied 90 min later. Chronic hemiorchidectomy significantly increased the serum testosterone response to LH at 17 but not at 24 days of age. The testicular response to LH in vitro was also investigated. Testes from rats hemiorchidectomized or sham-operated at 10 days of age and killed at 17 and 24 days of age were incubated for 3 h in the presence or absence of 100 ng LH/ml incubation medium. Significantly more 5α-androstane-3α,17β-diol and androsterone were produced by the testes from hemiorchidectomized than from sham-operated animals at 17 days of age but not at 24 days of age. The production of testosterone in vitro was not influenced by hemiorchidectomy at either age. These results suggest that serum FSH concentration is the factor limiting LH responsiveness before but not after the prepubertal increase in FSH levels. However, treatment of intact rats from 10 to 16 days of age with 20 μg rat FSH/day did not increase the serum testosterone response to LH.

Anti-viral CTL were induced in vitro using a particle bombardment device or "gene-gun" to deliver plasmid DNA encoding the nucleoprotein of the lymphocytic choriomeningitis virus (LCMV). Using this plasmid we were able to study T cell-mediated immunity in the absence of a neutralizing Ab response. Upon a single DNA immunization, a nearly 2 log10 reduction in splenic viral titers was observed 3 days after LCMV infection. After two or three immunizations a greater than 3 log10 inhibition of viral titers in the spleen was observed, with most animals having no detectable virus. C57BL/6 mice immunized with DNA encoding the nucleoprotein gene were also challenged with LCMV intracranially. Upon intracranial challenge, vaccinated animals displayed either protection or enhanced immunopathology leading to accelerated kinetics of death. Using limiting dilution analysis it was possible to detect LCMV-specific CTL precursors in both the spleen and lymph nodes of vaccinated animals. C57BL/6 mice inoculated with DNA demonstrated an anamnestic CTL response detectable at day 4 after LCMV challenge. Thus DNA vaccines are capable of inducing an anti-viral T cell response that can inhibit viral replication and mediate either protective immunity or immunopathology. Vaccination with DNA may therefore provide a useful alternative to current viral or subunit vaccines once the efficacy of immunization with DNA is optimized.

Anti-viral CTL were induced in vitro using a particle bombardment device or "gene-gun" to deliver plasmid DNA encoding the nucleoprotein of the lymphocytic choriomeningitis virus (LCMV). Using this plasmid we were able to study T cell-mediated immunity in the absence of a neutralizing Ab response. Upon a single DNA immunization, a nearly 2 log10 reduction in splenic viral titers was observed 3 days after LCMV infection. After two or three immunizations a greater than 3 log10 inhibition of viral titers in the spleen was observed, with most animals having no detectable virus. C57BL/6 mice immunized with DNA encoding the nucleoprotein gene were also challenged with LCMV intracranially. Upon intracranial challenge, vaccinated animals displayed either protection or enhanced immunopathology leading to accelerated kinetics of death. Using limiting dilution analysis it was possible to detect LCMV-specific CTL precursors in both the spleen and lymph nodes of vaccinated animals. C57BL/6 mice inoculated with DNA demonstrated an anamnestic CTL response detectable at day 4 after LCMV challenge. Thus DNA vaccines are capable of inducing an anti-viral T cell response that can inhibit viral replication and mediate either protective immunity or immunopathology. Vaccination with DNA may therefore provide a useful alternative to current viral or subunit vaccines once the efficacy of immunization with DNA is optimized.

T cell responses to viral infections can mediate either protective immunity or damaging immunopathology. Viral infections induce the proliferation of T cells specific for viral antigens and cause a loss in the number of T cells with other specificities. In immunologically naive hosts, viruses will induce T cell responses that, dependent on the MHC, recognize a distinct hierarchy of virus-encoded T cell epitopes. This hierarchy can change if the host has previously encountered another pathogen that elicited a memory pool ofT cells specific to a cross-reactive epitope. This heterologous immunity can deviate the normal immune response and result in either beneficial or harmful effects on the host. Each host has a unique T cell repertoire caused by the random DNA rearrangement that created it, so the specific T cells that create the epitope hierarchy differ between individuals. This "private specificity" seems of little significance in the T cell response of a naive host to infection, but it is of profound importance under conditions of heterologous immunity, where a small subset of a cross-reactive memory pool may expand and dominate a response. Examples are given of how the private specificities of immune responses under conditions of heterologous immunity influence the pathogenesis of murine and human viral infections.