Dengue virus-specific, human CD4+ CD8- cytotoxic T-cell clones: multiple patterns of virus cross-reactivity recognized by NS3-specific T-cell clones.

Thirteen dengue virus-specific, cytotoxic CD4+ CD8- T-cell clones were established from a donor who was infected with dengue virus type 3. These clones were examined for virus specificity and human leukocyte antigen (HLA) restriction in cytotoxic assays. Six patterns of virus specificities were determined. Two serotype-specific clones recognized only dengue virus type 3. Two dengue virus subcomplex-specific clones recognized dengue virus types 2, 3, and 4, and one subcomplex-specific clone recognized dengue virus types 1, 2, and 3. Four dengue virus serotype-cross-reactive clones recognized dengue virus types 1, 2, 3, and 4. One flavivirus-cross-reactive clone recognized dengue virus types 1, 2, 3, and 4 and West Nile virus (WNV), but did not recognize yellow fever virus (YFV), whereas three flavivirus-cross-reactive clones recognized dengue virus types 1, 2, 3, and 4, WNV, and YFV. HLA restriction in the lysis by these T-cell clones was also heterogeneous. HLA-DP, HLA-DQ, and HLA-DR were used as restriction elements by various T-cell clones. We also examined the recognition of viral nonstructural protein NS3, purified from cells infected with dengue virus type 3 or WNV, by these T-cell clones. One serotype-specific clone, two dengue virus subcomplex-specific clones, and three dengue virus serotype-cross-reactive clones recognized NS3 of dengue virus type 3. One flavivirus-cross-reactive clone recognized NS3 of dengue virus type 3 and WNV. These results indicate that heterogeneous dengue virus-specific CD4+ cytotoxic T cells are stimulated in response to infection with a dengue virus and that a nonstructural protein, NS3, contains multiple dominant T-cell epitopes.     

Analysis of Murine CD8+ T-Cell Clones Specific for the Dengue Virus NS3 Protein: Flavivirus Cross-Reactivity and Influence of Infecting Serotype

Serotype-cross-reactive dengue virus-specific cytotoxic T lymphocytes (CTL) induced during a primary dengue virus infection are thought to play a role in the immunopathogenesis of dengue hemorrhagic fever (DHF) during a secondary dengue virus infection. Although there is no animal model of DHF, we previously reported that murine dengue virus-specific CTL responses are qualitatively similar to human dengue virus-specific CTL responses. We used BALB/c mice to study the specificity of the CTL response to an immunodominant epitope on the dengue virus NS3 protein. We mapped the minimal H-2K^d-restricted CTL epitope to residues 298 to 306 of the dengue type 2 virus NS3 protein. In short-term T-cell lines and clones, the predominant CD8⁺ CTL to this epitope in mice immunized with dengue type 2 virus or vaccinia virus expressing the dengue type 4 virus NS3 protein were cross-reactive with dengue type 2 or type 4 virus, while broadly serotype-cross-reactive CTL were a minority population. In dengue type 3 virus-immunized mice, the predominant CTL response to this epitope was broadly serotype cross-reactive. All of the dengue virus-specific CTL clones studied also recognized the homologous NS3 sequences of one or more closely related flaviviruses, such as Kunjin virus. The critical contact residues for the CTL clones with different specificities were mapped with peptides having single amino acid substitutions. These data demonstrate that primary dengue virus infection induces a complex population of flavivirus-cross-reactive NS3-specific CTL clones in mice and suggest that CTL responses are influenced by the viral serotype. These findings suggest an additional mechanism by which the order of sequential flavivirus infections may influence disease manifestations.     

Multiple specificities in the murine CD4+ and CD8+ T-cell response to dengue virus.

The target epitopes, serotype specificity, and cytolytic function of dengue virus-specific T cells may influence their theoretical roles in protection against secondary infection as well as the immunopathogenesis of dengue hemorrhagic fever. To study these factors in an experimental system, we isolated dengue virus-specific CD4+ and CD8+ T-cell clones from dengue-2 virus-immunized BALB/c mice. The T-cell response to dengue virus in this mouse strain was heterogeneous; we identified at least five different CD4+ phenotypes and six different CD8+ phenotypes. Individual T-cell clones recognized epitopes on the dengue virus pre-M, E, NSl/NS2A, and NS3 proteins and were restricted by the I-Ad, I-Ed, Ld, and Kd antigens. Both serotype-specific and serotype-cross-reactive clones were isolated in the CD4+ and CD8+ subsets; among CD8+ clones, those that recognized the dengue virus structural proteins were serotype specific whereas those that recognized the nonstructural proteins were serotype cross-reactive. All of the CD8+ and one of five CD4+ clones lysed dengue virus-infected target cells. Using synthetic peptides, we identified an Ld-restricted epitope on the E protein (residues 331 to 339, SPCKIPFEI) and a Kd-restricted epitope on the NS3 protein (residues 296 to 310, ARGYISTRVEM GEAA). These data parallel previous findings of studies using human dengue virus-specific T-cell clones. This experimental mouse system may be useful for studying the role of the virus serotype and HLA haplotype on T-cell responses after primary dengue virus infection.     

Predominance of HLA-Restricted Cytotoxic T-Lymphocyte Responses to Serotype-Cross-Reactive Epitopes on Nonstructural Proteins following Natural Secondary Dengue Virus Infection

We examined the memory cytotoxic T-lymphocytic (CTL) responses of peripheral blood mononuclear cells (PBMC) obtained from patients in Thailand 12 months after natural symptomatic secondary dengue virus infection. In all four patients analyzed, CTLs were detected in bulk culture PBMC against nonstructural dengue virus proteins. Numerous CD4⁺ and CD8⁺ CTL lines were generated from the bulk cultures of two patients, KPP94-037 and KPP94-024, which were specific for NS1.2a (NS1 and NS2a collectively) and NS3 proteins, respectively. All CTL lines derived from both patients were cross-reactive with other serotypes of dengue virus. The CD8⁺ NS1.2a-specific lines from patient KPP94-037 were HLA B57 restricted, and the CD8⁺ NS3-specific lines from patient KPP94-024 were HLA B7 restricted. The CD4⁺ CTL lines from patient KPP94-037 were HLA DR7 restricted. A majority of the CD8⁺ CTLs isolated from patient KPP94-024 were found to recognize amino acids 221 to 232 on NS3. These results demonstrate that in Thai patients after symptomatic secondary natural dengue infections, CTLs are mainly directed against nonstructural proteins and are broadly cross-reactive.     

Dengue virus-specific human CD4+ T-lymphocyte responses in a recipient of an experimental live-attenuated dengue virus type 1 vaccine: bulk culture proliferation, clonal analysis, and precursor frequency determination.

We analyzed the CD4+ T-lymphocyte responses to dengue, West Nile, and yellow fever viruses 4 months after immunization of a volunteer with an experimental live-attenuated dengue virus type 1 vaccine (DEN-1 45AZ5). We examined bulk culture proliferation to noninfectious antigens, determined the precursor frequency of specific CD4+ T cells by limiting dilution, and established and analyzed CD4+ T-cell clones. Bulk culture proliferation was predominantly dengue virus type 1 specific with a lesser degree of cross-reactive responses to other dengue virus serotypes, West Nile virus, and yellow fever virus. Precursor frequency determination by limiting dilution in the presence of noninfectious dengue virus antigens revealed a frequency of antigen-reactive cells of 1 in 1,686 peripheral blood mononuclear cells (PBMC) for dengue virus type 1, 1 in 9,870 PBMC for dengue virus type 3, 1 in 14,053 PBMC for dengue virus type 2, and 1 in 17,690 PBMC for dengue virus type 4. Seventeen CD4+ T-cell clones were then established by using infectious dengue virus type 1 as antigen. Two patterns of dengue virus specificity were found in these clones. Thirteen clones were dengue virus type 1 specific, and four clones recognized both dengue virus types 1 and 3. Analysis of human leukocyte antigen (HLA) restriction revealed that five clones are HLA-DRw52 restricted, one clone is HLA-DP3 restricted, and one clone is HLA-DP4 restricted. These results indicate that in this individual, the CD4+ T-lymphocyte responses to immunization with live-attenuated dengue virus type 1 vaccine are predominantly serotype specific and suggest that a multivalent vaccine may be necessary to elicit strong serotype-cross-reactive CD4+ T-lymphocyte responses in such individuals.     

Bystander target cell lysis and cytokine production by dengue virus-specific human CD4(+) cytotoxic T-lymphocyte clones

Dengue hemorrhagic fever, the severe form of dengue virus infection, is believed to be an immunopathological response to a secondary infection with a heterologous serotype of dengue virus. Dengue virus capsid protein-specific CD4(+) cytotoxic T-lymphocyte (CTL) clones were shown to be capable of mediating bystander lysis of non-antigen-presenting target cells. After activation by anti-CD3 or in the presence of unlabeled antigen-presenting target cells, these clones could lyse both Jurkat cells and HepG2 cells as bystander targets. Lysis of HepG2 cells suggests a potential role for CD4(+) CTL in the liver involvement observed during dengue virus infection. Three CD4(+) CTL clones were demonstrated to lyse cognate, antigen-presenting target cells by a mechanism that primarily involves perforin, while bystander lysis occurred through Fas/Fas ligand interactions. In contrast, one clone used a Fas/Fas ligand mechanism to lyse both cognate and bystander targets. Cytokine production by the CTL clones was also examined. In response to stimulation with D2 antigen, CD4(+) T-cell clones produced gamma interferon, tumor necrosis factor alpha (TNF-alpha) and TNF-beta. The data suggest that CD4(+) CTL clones may contribute to the immunopathology observed upon secondary dengue virus infections through direct cytolysis and/or cytokine production.     

Differential functional avidity of dengue virus-specific T-cell clones for variant peptides representing heterologous and previously encountered serotypes

Proinflammatory cytokines secreted by memory CD8+ and CD4+ T cells are thought to play a direct role in the pathogenesis of dengue virus infection by increasing vascular permeability and thereby inducing the pathophysiologic events associated with dengue hemorrhagic fever and dengue shock syndrome. Severe disease is frequently observed in the setting of secondary infection with heterologous dengue virus serotypes, suggesting a role for cross-reactive memory T cells in the immunopathogenesis of severe disease. We used a large panel of well-characterized dengue virus-specific CD8+ T-cell clones isolated from Pacific Islanders previously infected with dengue virus 1 to examine effector memory function, focusing on a novel dominant HLA-B*5502-restricted NS5(329-337) epitope, and assessed T-cell responses to stimulation with variant peptides representing heterologous serotypes. Variant peptides were differentially recognized by dengue virus 1-specific effector CD8+ cytotoxic T lymphocytes (CTL) in a heterogeneous and clone-specific manner, in which cytolytic function and cytokine secretion could be enhanced, diminished, or abrogated compared with cognate peptide stimulation. Dengue virus-specific CTL stimulated with cognate and variant peptides demonstrated a cytokine response hierarchy of gamma IFN (IFN-gamma) > tumor necrosis factor alpha (TNF-alpha) > interleukin-2 (IL-2), and a subset of clones also produced IL-4 and IL-6. Individual clones demonstrated greater avidity for variant peptides representing heterologous serotypes, including serotypes previously encountered by the subject, and IFN-gamma and TNF-alpha secretion was enhanced by stimulation with these heterologous peptides. Altered antiviral T-cell responses in response to stimulation with heterologous dengue virus serotypes have implications for control of virus replication and for disease pathogenesis.     

Identification of a naturally processed cytotoxic CD8 T-cell epitope of coxsackievirus B4, presented by HLA-A2.1 and located in the PEVKEK region of the P2C nonstructural protein

The adaptive immune system generates CD8 cytotoxic T lymphocytes (CTLs) as a major component of the protective response against viruses. Knowledge regarding the nature of the peptide sequences presented by HLA class I molecules and recognized by CTLs is thus important for understanding host-pathogen interactions. In this study, we focused on identification of a CTL epitope generated from coxsackievirus B4 (CVB4), a member of the enterovirus group responsible for several inflammatory diseases in humans and often implicated in the triggering and/or acceleration of the autoimmune disease type 1 diabetes. We identified a 9-mer peptide epitope that can be generated from the P2C nonstructural protein of CVB4 (P2C(1137-1145)) and from whole virus by antigen-presenting cells and presented by HLA-A2.1. This epitope is recognized by effector memory (gamma interferon [IFN-gamma]-producing) CD8 T cells in the peripheral blood at a frequency of responders that suggests that it is a major focus of the anti-CVB4 response. Short-term CD8 T-cell lines generated against P2C(1137-1145) are cytotoxic against peptide-loaded target cells. Of particular interest, the epitope lies within a region of viral homology with the diabetes-related autoantigen, glutamic acid decarboxylase-65 (GAD(65)). However, P2C(1137-1145)-specific cytotoxic T lymphocyte (CTL) lines were not activated to produce IFN-gamma by the GAD(65) peptide homologue and did not show cytotoxic activity in the presence of appropriately labeled targets. These results describe the first CD8 T-cell epitope of CVB4 that will prove useful in the study of CVB4-associated disease.     

Vaccinia virus-specific CD8+ cytotoxic T lymphocytes in humans.

Stimulation of human vaccinia virus immune peripheral blood mononuclear cells in vitro from vaccinia virus-immune donors with live vaccinia virus-infected autologous cells generated vaccinia virus-specific cytotoxic T lymphocytes (CTL) capable of lysing vaccinia virus-infected cells. We generated vaccinia virus-specific CD8+ clones and CD4+ CTL lines by limiting dilution from two donors by using peripheral blood mononuclear cells obtained 2 months or 4 years postrevaccination with vaccinia virus. These results demonstrate that vaccinia virus-specific CTL are generated as a result of immunization of humans with vaccinia virus and that both CD8(+)- and CD4(+)-specific T cells are maintained as memory cells.     

Delayed clearance of Sendai virus in mice lacking class I MHC-restricted CD8+ T cells.

The role and interdependence of CD8+ and CD4+ alpha beta-T cells in the acute response after respiratory infection with the murine parainfluenza type 1 virus, Sendai virus, has been analyzed for H-2b mice. Enrichment of CD8+ virus-specific CTL effectors in the lungs of immunologically intact C57BL/6 animals coincided with the clearance of the virus from this site by day 10 after infection. Removal of the CD4+ T cells by in vivo mAb treatment did not affect appreciably either the recruitment of CD8+ T cells to the infected lung, or their development into virus-specific cytotoxic effectors. In contrast, depletion of the CD8+ subset delayed virus clearance, although most mice survived the infection. Transgenic H-2b F3 mice homozygous (-/-) for a beta 2 microglobulin (beta 2-m) gene disruption, which lack both class I MHC glycoproteins and mature CD8+ alpha beta-T cells, showed a comparable, delayed clearance of Sendai virus from the lung. Virus-specific, class II MHC-restricted CTL were demonstrated in both freshly isolated bronchoalveolar lavage populations and cultured lymph node and spleen tissue from the beta 2-m (-/-) transgenics. Treatment of the beta 2-m (-/-) mice with the mAb to CD4 led to delayed virus clearance and death, which was also the case for normal mice that were depleted simultaneously of the CD4+ and CD8+ subsets. These results indicate that, although classical class I MHC-restricted CD8+ cytotoxic T cells normally play a dominant role in the recovery of mice acutely infected with Sendai virus, alternative mechanisms involving CD4+ T cells exist and can compensate, in time, for the loss of CD8+ T cell function.     

Composite response of naive T cells to stimulation with the autologous lymphoblastoid cell line is mediated by CD4 cytotoxic T cell clones and includes an Epstein-Barr virus-specific component.

We have approached the challenge of generating a primary T cell response to Epstein-Barr virus (EBV) in vitro by stimulating naive T cells with the autologous EBV-transformed lymphoblastoid cell line (LCL), a rich source of EBV-associated cytotoxic T lymphocyte (CTL) epitopes. Responsive T cells from three EBV-seronegative donors were cloned in agarose, phenotyped for T cell markers by flow cytometry, and their cytotoxic properties analyzed in the 51Cr release assay. Most clones (greater than 95%) expressed the CD4 phenotype and 59% of these clones showed cytotoxic properties. The dominant CTL response was specific for FCS-associated epitopes presented by FCS-grown autologous LCL target cells and was restricted by class II HLA antigens. Other clonal components included: (i) an EBV-specific response by HLA-restricted CD4 CTL clones that did not discriminate between A- and B-type EBV transformants; (ii) an EBV-specific response by an HLA-restricted CD4 CTL clone that discriminated between A- and B-type transformants, and (iii) a nonspecific cytotoxic response by CD3+,4+,8-, CD3+,4-,8-, and CD3-,4-,8- clones that were broadly allotypic or restricted to the lysis of K562 target cells. The EBV-specific CTL clones did not lyse the autologous EBV-negative B or T cell blasts and their specificity patterns of lysis were supported by the cold target competition data. These studies highlight the role of CD4 CTL in the establishment in vitro of a primary immune response to a human virus.     

A CD4+ cytotoxic T-lymphocyte clone to a conserved epitope on human immunodeficiency virus type 1 p24: cytotoxic activity and secretion of interleukin-2 and interleukin-6.

A CD4+ cytotoxic T-lymphocyte (CTL) clone, established from the peripheral blood of a human immunodeficiency virus (HIV)-seropositive donor, lysed autologous target cells that were infected with a recombinant vaccinia virus containing the gag gene of HIV type 1 and target cells pulsed with p24gag construct expressed in Escherichia coli. The recognition of the HLA-DQ-restricted epitope by this clone was further defined by using overlapping synthetic peptides. The epitope recognized by this CD4+ CTL clone (amino acids 140 to 148) overlaps with a CD8+ epitope and is highly conserved among all isolates of HIV type 1 that have been sequenced. Production and secretion of lymphokines such as interleukin-2 and interleukin-6 after specific antigenic stimulation were demonstrated by this gag-specific CD4+ CTL clone.     

Characterization of mouse hepatitis virus-specific cytotoxic T cells derived from the central nervous system of mice infected with the JHM strain.

The cytotoxic T lymphocyte (CTL) activity of spleen cells from BALB/c (H-2d) mice immunized with the neurotropic JHM strain of mouse hepatitis virus (JHMV) was stimulated in vitro for 7 days. CTL were tested for recognition of target cells infected with either JHMV or vaccinia virus recombinants expressing the four virus structural proteins. Only target cells infected with either JHMV or the vaccinia virus recombinant expressing the JHMV nucleocapsid protein were recognized. Cytotoxic T cell lines were established by limiting dilution from the brains of mice undergoing acute demyelinating encephalomyelitis after infection with JHMV. Twenty of the 22 lines recognized JHMV-infected but not uninfected syngeneic target cells, indicating that they are specific for JHMV. All T-cell lines except one were CD8+. The specificity of the CTL lines was examined by using target cells infected with vaccinia virus recombinants expressing the JHMV nucleocapsid, spike, membrane, and hemagglutinin-esterase structural proteins. Seventeen lines recognized target cells expressing the nucleocapsid protein. Three of the JHMV-specific T-cell lines were unable to recognize target cells expressing any of the JHMV structural proteins, indicating that they are specific for an epitope of a nonstructural protein(s) of JHMV. These data indicate that the nucleocapsid protein induces an immunodominant CTL response. However, no CTL activity specific for the nucleocapsid protein could be detected in either the spleens or cervical lymph nodes of mice 4, 5, 6, or 7 days after intracranial infection, suggesting that the CTL response to JHMV infection within the central nervous system may be induced or expanded locally.     

Antiviral protection by CD8+ versus CD4+ T cells. CD8+ T cells correlating with cytotoxic activity in vitro are more efficient in antivaccinia virus protection than CD4-dependent IL.

T cell-mediated protection against a recombinant vaccinia virus was evaluated in mice with respect to the relative contributions of CTL vs that of T cell-dependent IL and of CD4+ cells. H-2b mice primed with the wildtype of vesicular stomatitis virus serotype Indiana (VSV-IND wt) mount an in vitro measurable cytotoxic response against the nucleoprotein (NP) of VSV-IND and are protected against a challenge infection with a vaccinia-VSV recombinant virus expressing the NP of VSV-IND (vacc-IND-NP). Their protective mechanism was highly susceptible to in vivo depletion of CD8+ T cells, but resistant to CD4+ depletion or treatment with anti-IFN-gamma and anti-TNF-alpha. Surprisingly, also VSV-CTL nonresponder H-2k mice were protected against a challenging infection with vacc-IND-NP when primed with VSV-IND wt. In contrast to the CTL responder H-2b mice, this protection was highly susceptible to CD4+ T cell depletion and to anti-IFN-gamma or anti-TNF-alpha treatment, but resistant to CD8+ T cell depletion. Antibodies were not responsible because they failed to transfer protection; in contrast CD4+ T cells conferred significant protection. VSV-CTL responder H-2b and nonresponder H-2k mice were protected almost equally well against a challenge dose of 10(3) pfu vacc-IND-NP inoculated intracerebrally. However, after intracerebral challenge with 5 x 10(6) pfu vacc-IND-NP, the CTL nonresponder mice died, whereas the CTL responder mice eliminated the virus by day 5. These results collectively show that CD4+ T cell-dependent IL may mediate antiviral protection, but their efficiency is relatively weak compared with CD8-mediated protection correlating with cytotoxic activity in vitro.     

Identification of amino acids involved in recognition by dengue virus NS3-specific, HLA-DR15-restricted cytotoxic CD4+ T-cell clones.

The majority of T-cell clones derived from a donor who experienced dengue illness following receipt of a live experimental dengue virus type 3 (DEN3) vaccine cross-reacted with all four serotypes of dengue virus, but some were serotype specific or only partially cross-reactive. The nonstructural protein, NS3, was immuno-dominant in the CD4+ T-cell response of this donor. The epitopes of four NS3-specific T-cell clones were analyzed. JK15 and JK13 recognized only DEN3 NS3, while JK44 recognized DEN1, DEN2, and DEN3 NS3 and JK5 recognized DEN1, DEN3, and West Nile virus NS3. The epitopes recognized by these clones on the DEN3 NS3 protein were localized with recombinant vaccinia viruses expressing truncated regions of the NS3 gene, and then the minimal recognition sequence was mapped with synthetic peptides. Amino acids critical for T-cell recognition were assessed by using peptides with amino acid substitutions. One of the serotype-specific clones (JK13) and the subcomplex- and flavivirus-cross-reactive clone (JK5) recognized the same core epitope, WITDFVGKTVW. The amino acid at the sixth position of this epitope is critical for recognition by both clones. Sequence analysis of the T-cell receptors of these two clones showed that they utilize different VP chains. The core epitopes for the four HLA-DR15-restricted CD4+ CTL clones studied do not contain motifs similar to those proposed by previous studies on endogenous peptides eluted from HLA-DR15 molecules. However, the majority of these dengue virus NS3 core epitopes have a positive amino acid (K or R) at position 8 or 9. Our results indicate that a single epitope can induce T cells with different virus specificities despite the restriction of these T cells by the same HLA-DR15 allele. This finding suggests a previously unappreciated level of complexity for interactions between human T-cell receptors and viral epitopes with very similar sequences on infected cells.     

Different effects of Nef-mediated HLA class I down-regulation on human immunodeficiency virus type 1-specific CD8(+) T-cell cytolytic activity and cytokine production

A previous study using a Nef-defective human immunodeficiency virus type 1 (HIV-1) mutant suggested that Nef-mediated down-regulation of HLA class I on the infected cell surface affects the cytolytic activity of HIV-1-specific cytotoxic T-lymphocyte (CTL) clones for HIV-1-infected primary CD4(+) T cells. We confirmed this effect by using a nef-mutant HIV-1 strain (NL-M20A) that expresses a Nef protein which does not induce down-regulation of HLA class I molecules but is otherwise functional. HIV-1-specific CTL clones were not able to kill primary CD4(+) T cells infected with a Nef-positive HIV-1 strain (NL-432) but efficiently lysed CD4(+) T cells infected with NL-M20A. Interestingly, CTL clones stimulated with NL-432-infected CD4(+) T cells were able to produce cytokines, albeit at a lower level than when stimulated with NL-M20A-infected CD4(+) T cells. This indicates that Nef-mediated HLA class I down-regulation affects CTL cytokine production to a lesser extent than cytolytic activity. Replication of NL-432 was partially suppressed in a coculture of HIV-1-infected CD4(+) T cells and HIV-1-specific CTL clones, while replication of NL-M20A was completely suppressed. These results suggest that HIV-1-specific CD8(+) T cells are able to partially suppress the replication of HIV-1 through production of soluble HIV-1-suppressive factors such as chemokines and gamma interferon. These findings may account for the mechanism whereby HIV-1-specific CD8(+) T cells are able to partially but not completely control HIV-1 replication in vivo.     

Expression of dengue virus structural proteins and nonstructural protein NS1 by a recombinant vaccinia virus.

A recombinant vaccinia virus containing cloned DNA sequences coding for the three structural proteins and nonstructural proteins NS1 and NS2a of dengue type 4 virus was constructed. Infection of CV-1 cells with this recombinant virus produced dengue virus structural proteins as well as the nonstructural protein NS1. These proteins were precipitated by specific antisera and exhibited the same molecular size and glycosylation patterns as authentic dengue virus proteins. Infection of cotton rats with the recombinant virus induced NS1 antibodies in 1 of 11 animals. However, an immune response to the PreM and E glycoproteins was not detected. A reduced level of gene expression was probably the reason for the limited serologic response to these dengue virus antigens.     

CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection.

In this study, we have examined the relative contributions of CD4+ and CD8+ T cells in controlling an acute or chronic lymphocytic choriomeningitis virus (LCMV) infection. To study acute infection, we used the LCMV Armstrong strain, which is cleared by adult mice in 8 to 10 days, and to analyze chronic infection, we used a panel of lymphocyte-tropic and macrophage-tropic variants of LCMV that persist in adult mice for several months. We show that CD4+ T cells are not necessary for resolving an acute LCMV infection. CD4+ T-cell-depleted mice were capable of generating an LCMV-specific CD8+ cytotoxic T-lymphocyte (CTL) response and eliminated virus with kinetics similar to those for control mice. The CD8+ CTL response was critical for resolving this infection, since beta 2-microglobulin knockout (CD8-deficient) mice were unable to control the LCMV Armstrong infection and became persistently infected. In striking contrast to the acute infection, even a transient depletion of CD4+ T cells profoundly affected the outcome of infection with the macrophage- and lymphocyte-tropic LCMV variants. Adult mice given a single injection of anti-CD4 monoclonal antibody (GK1.5) at the time of virus challenge became lifelong carriers with high levels of virus in most tissues. Unmanipulated adult mice infected with the different LCMV variants contained virus for prolonged periods (> 3 months) but eventually eliminated infection from most tissues, and all of these mice had LCMV-specific CD8+ CTL responses. Although the level of CTL activity was quite low, it was consistently present in all of the chronically infected mice that eventually resolved the infection. These results clearly show that even in the presence of an overwhelming viral infection of the immune system, CD8+ CTL can remain active for long periods and eventually resolve and/or keep the virus infection in check. In contrast, LCMV-specific CTL responses were completely lost in chronically infected CD4-depleted mice. Taken together, these results show that CD4+ T cells are dispensable for short-term acute infection in which CD8+ CTL activity does not need to be sustained for more than 2 weeks. However, under conditions of chronic infection, in which CD8+ CTLs take several months or longer to clear the infection, CD4+ T-cell function is critical. Thus, CD4+ T cells play an important role in sustaining virus-specific CD8+ CTL during chronic LCMV infection. These findings have implications for chronic viral infections in general and may provide a possible explanation for the loss of human immunodeficiency virus-specific CD8+ CTL activity that is seen during the late stages of AIDS, when CD4+ T cells become limiting.     

Patterns of cytokine production in human immunodeficiency virus type 1 (HIV-1)-specific human CD8+ T cells after stimulation with HIV-1-infected CD4+ T cells

Although human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells can produce various cytokines that suppress HIV-1 replication or modulate anti-HIV-1 immunity, the extent to which HIV-1-specific CD8+ T cells produce cytokines when they recognize HIV-1-infected CD4+ T cells in vivo still remains unclear. We first analyzed the abilities of 10 cytotoxic T-lymphocyte (CTL) clones specific for three HIV-1 epitopes to produce gamma interferon, macrophage inflammatory protein 1beta, and tumor necrosis factor alpha after stimulation with epitope peptide-pulsed cells. These CTL clones produced these cytokines in various combinations within the same specificity and among the different specificities, suggesting a functional heterogeneity of HIV-1-specific effector CD8+ T cells in cytokine production. In contrast, the HIV-1-specific CTL clones for the most part produced a single cytokine, without heterogeneity of cytokine production among the clones, after stimulation with HIV-1-infected CD4+ T cells. The loss of heterogeneity in cytokine production may be explained by low surface expression of HLA class I-epitope peptide complexes. Freshly isolated HIV-1-specific CD8+ T cells with an effector/memory or memory phenotype produced much more of the cytokines than the same epitope-specific CTL clones when stimulated with HIV-1-infected CD4+ T cells. Cytokine production from HIV-1-specific memory/effector and memory CD8+ T cells might be a critical event in the eradication of HIV-1 in HIV-1-infected individuals.     

Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome.

Virologic and immunologic studies were performed on five patients presenting with primary human immunodeficiency virus type 1 (HIV-1) infection. CD8+ cytotoxic T lymphocyte (CTL) precursors specific for cells expressing antigens of HIV-1 Gag, Pol, and Env were detected at or within 3 weeks of presentation in four of the five patients and were detected in all five patients by 3 to 6 months after presentation. The one patient with an absent initial CTL response had prolonged symptoms, persistent viremia, and low CD4+ T-cell count. Neutralizing antibody activity was absent at the time of presentation in all five patients. These findings suggest that cellular immunity is involved in the initial control of virus replication in primary HIV-1 infection and indicate a role for CTL in protective immunity to HIV-1 in vivo.