Long term persistence of herpes simplex virus-specific CD8+ CTL in persons with frequently recurring genital herpes

Herpes simplex virus (HSV) establishes a lifelong infection in humans. Reactivation of latent virus occurs intermittently so that the immune system is frequently exposed to viral Ag, providing an opportunity to evaluate memory T cells to a persistent human pathogen. We studied the persistence of genital herpes lesion-derived HSV-specific CD8+ CTL from three immunocompetent individuals with frequently recurring genital HSV-2 infection. All CTL clones were HSV-2 type specific and only one to three unique clonotypes were identified from any single biopsy specimen. The TCRBV genes utilized by these clonotypes were sequenced, and clonotype-specific probes were used to longitudinally track these clonotypes in PBMC and genital lesions. CTL clonotypes were consistently detected in PBMC and lesions for at least 2 and up to 7 years, and identical clonotypes infiltrated herpes lesions spaced as long as 7.5 years apart. Moreover, these clones were functionally lytic in vivo over these time periods. Additionally, CTL clones killed target cells infected with autologous viral isolates obtained 6.5 years after CTL clones were established, suggesting that selective pressure by these CTL did not result in the mutation of CTL epitopes. Thus, HSV recurs in the face of persistent CD8+ CTL with no evidence of clonal exhaustion or mutation of CTL epitopes as mechanisms of viral persistence.     

Diversity of the CD8+ T-cell response to herpes simplex virus type 2 proteins among persons with genital herpes

Cytolytic T cells play a major role in controlling herpes simplex virus type 2 (HSV-2) infections in humans. In an effort to more thoroughly evaluate the response to HSV-2 directly, ex vivo, we developed an enzyme-linked immunospot (ELISPOT) assay that utilized pools of overlapping synthetic peptides presented by autologous dendritic cells to purified CD8(+) T cells. Donor response rates to individual open reading frames (ORFs) ranged from fewer than 5% responding to as many as 70% responding, with the greatest frequency of responses (by ORF) being directed against UL39, UL25, UL27, ICP0, UL46, and UL47 in descending order of frequency. HSV-2-seropositive subjects responded to as few as 3 or as many as 46 of the 48 ORFs tested, with a median of 11 ORFs recognized. HLA-B*07 expression correlated with stronger responses overall that were directed primarily against UL49 and UL46. Cumulative precursor frequencies in the blood ranged from 500 to almost 6,000 HSV-2 spot-forming units/10(6) CD8(+) T cells. The magnitude and breadth of the response in the infected population were greater than previously appreciated. Whether this variability in the CD8(+) T-cell response within individuals is associated with the frequency of viral reactivation warrants further study.     

Characteristics of chromatin preparations from herpes simplex virus infected cells

Chromatin isolated from herpes simplex virus type 1-infected baby hamster kidney cells contains a number of tightly associated virus-induced polypeptides. A subset of these proteins bind to immobilized DNA in vitro (Vmw 175, 155, 130, 63, 43, 38/39). Virus-induced polypeptides extractable with acid from infected cell chromatin include Vmw 155, the major capsid protein of herpes simplex virus type 1 virions, and Vmw 63 and 38/39 which are heterogeneous with respect to charge and are phosphorylated. These chromatin preparations, in the presence of deoxynucleoside triphosphates and MgCl2 were capable of synthesizing viral and cell DNA in a reaction which was stimulated by the addition of ATP, riboNTPs and potassium acetate. In vitro synthesized viral DNA co-sedimented with prelabelled parental DNA but the single-stranded product was smaller than parental DNA. Density labelling indicated that extensive synthesis was taking place and all BamHI fragments of viral DNA were represented by the DNA synthesized in vitro.     

HLA-restricted lysis of herpes simplex virus-infected monocytes and macrophages mediated by CD4+ and CD8+ T lymphocytes

Freshly isolated human peripheral blood monocytes and in vitro monocyte-derived macrophages were infected with HSV type 1 and used as target cells in a cell-mediated cytotoxicity assay. PBMC from both HSV-immune and non-immune donors were stimulated in vitro for 5 days with UV-inactivated HSV Ag and used as effector cells. Effectors from HSV-immune donors mediated virus-specific lysis of both monocyte and macrophage targets, whereas effectors from non-immune donors failed to mediate target cell lysis. Mean virus-specific lysis of autologous monocytes was (8.5 +/- (+/- 2.0)%) compared to a threefold greater virus-specific lysis of autologous macrophages (24.7 (+/- 4.3)%). More than 70% of this lysis was mediated by CD16- T lymphocytes. Further analysis demonstrated that the majority of the lysis against autologous and allogeneic targets was HLA-DR-restricted and mediated by CD4+ CTL. However, CD8+ CTL also contributed to the lysis of autologous targets as well as allogeneic targets having a common HLA-A and/or -B determinant. The HLA-restricted cytotoxicity was virus-specific as HSV-infected, but not CMV-infected, cells were lysed. CTL-mediated lysis of HSV-infected monocytes and macrophages may be of significance in the anti-viral and immunoregulatory host response.     

The early expression of glycoprotein B from herpes simplex virus can be detected by antigen-specific CD8+ T cells

The immune response to cutaneous herpes simplex virus type 1 (HSV-1) infection begins with remarkable rapidity. Activation of specific cytotoxic T lymphocytes (CTL) begins within hours of infection, even though the response within the draining lymph nodes peaks nearly 5 days later. HSV gene products are classified into three main groups, alpha, beta, and gamma, based on their kinetics and requirements for expression. In C57BL/6 mice, the immunodominant epitope from HSV is derived from glycoprotein B (gB(498-505)). While gB is considered a gamma or "late" gene product, previous reports have indicated that some level of gene expression may occur soon after infection. Using brefeldin A as a specific inhibitor of viral antigen presentation to major histocompatibility complex class I-restricted CTL, we have formally addressed the timing of gB peptide expression in an immunologically relevant manner following infection. Presentation of gB peptide detected by T-cell activation was first observed within 2 h of infection. Comparison with another viral epitope expressed early during infection, HSV-1 ribonucleotide reductase, demonstrated that gB is presented with the same kinetics as this classical early-gene product. Moreover, this rapidity of gB expression was further illustrated via rapid priming of na?ve transgenic CD8(+) T cells in vivo after HSV-1 infection of mice. These results establish that gB is expressed rapidly following HSV-1 infection, at levels capable of effectively stimulating CD8(+) T cells.     

Phenotypic identification of antigen-dependent and antigen-independent CD8 CTL precursors in the draining lymph node during acute cutaneous herpes simplex virus type 1 infection.

Optimal immunological control of cutaneous herpes simplex virus type 1 (HSV-1) infections initiated in the hind footpad of C57BL/6 (B6, H-2b) mice is dependent upon the presence of functional HSV-1-specific T lymphocytes. The class I MHC-restricted, CD8+ T cell subpopulation is involved in the clearance of infectious HSV-1 from the skin and limiting HSV-1 replication and spread within the peripheral nervous system. However, the frequency of HSV-1-specific CTL precursors (CTLp), as a measure of potential anti-viral CD8+ T cell function, is relatively low compared with other acute viral infections. To gain insight into the basis for this low functional frequency, changes in the CD8+ T cell subpopulation phenotype associated with activation and differentiation were investigated. Analysis of the phenotypic changes showed that HSV-1-specific CTLp were found predominantly within a subpopulation of CD8+ T cells expressing high levels of CD44 (CD44high) and high levels of the IL-2 receptor alpha-chain (CD25high). A second activated subpopulation of CD8+ T cells expressing the CD44high CD25low phenotype did not contain detectable HSV-1-specific CTLp, even after the addition of HSV-1-infected stimulator cells as a source of an exogenous Ag. These data suggested that HSV-1-specific CD8+ T cells must increase expression of CD25 before attaining the potential to become CTL effector cells. These findings also indicated that the up-regulation of CD44 alone is not sufficient to identify precisely HSV-1-specific CD8+ T cells.     

CD8(+) T-cell attenuation of cutaneous herpes simplex virus infection reduces the average viral copy number of the ensuing latent infection

During an initial encounter with herpes simplex virus type 1 (HSV-1) it takes several days for an adaptive immune response to develop and for herpes-specific CD8(+) T cells to infiltrate sites of infection. By this time the virus has firmly established itself within the innervating sensory nervous system where it then persists indefinitely. Preventing the establishment of viral latency would require blocking the skin to nervous system transmission of the virus. We wished to examine if CD8(+) T cells present early during acute HSV-1 infection could block this transmission. We show that effector CD8(+) T cells failed to prevent the establishment of HSV latency even when present prior to infection. This lack of blocking likely reflects the delayed infiltration of the CD8(+) T cells into the infected skin. Examination of the kinetics of HSV-1 infection highlighted the rapidity at which the virus infects the sensory ganglia and singled out early viral replication within the skin as an important factor in determining the magnitude of the ensuing latent infection. Though unable to prevent the establishment of latency, CD8(+) T cells could reduce the average viral copy number of the residual latent infection by dampening the skin infection and thus limiting the skin-to-nerve transmission of virus.     

Antigenic specificities of human CD4+ T-cell clones recovered from recurrent genital herpes simplex virus type 2 lesions.

Lesions resulting from recurrent genital herpes simplex virus (HSV) infection are characterized by infiltration of CD4+ lymphocytes. We have investigated the antigenic specificity of 47 HSV-specific CD4+ T-cell clones recovered from the HSV-2 buttock and thigh lesions of five patients. Clones with proliferative responses to recombinant truncated glycoprotein B (gB) or gD of HSV-2 or purified natural gC of HSV-2 comprised a minority of the total number of HSV-specific clones isolated from lesions. The gC2- and gD2-specific CD4+ clones had cytotoxic activity. The approximate locations of the HSV-2 genes encoding HSV-2 type-specific CD4+ antigens have been determined by using HSV-1 x HSV-2 intertypic recombinant virus and include the approximate map regions 0.30 to 0.46, 0.59 to 0.67, 0.67 to 0.73, and 0.82 to 1.0 units. The antigenic specificity of an HLA DQ2-restricted, HSV-2 type-specific T-cell clone was mapped to amino acids 425 to 444 of VP16 of HSV-2 by sequential use of an intertypic recombinant virus containing VP16 of HSV-2 in an HSV-1 background, recombinant VP16 fusion proteins, and synthetic peptides. Each of the remaining four patients also yielded at least one type-specific T-cell clone reactive with an HSV-2 epitope mapping to approximately 0.67 to 0.73 map units. The antigenic specificities of lesion-derived CD4+ T-cell clones are quite diverse and include at least 10 epitopes. Human T-cell clones reactive with gC and VP16 are reported here for the first time.     

Cutting edge: conventional CD8 alpha+ dendritic cells are preferentially involved in CTL priming after footpad infection with herpes simplex virus-1

CTL play a major role in immunity to HSV type 1, but little is known about the priming process. In this study, we have examined the class I-restricted presentation of an immunodominant determinant from HSV-1 glycoprotein B after footpad infection. We have found that the only cell types capable of presenting this determinant in draining popliteal lymph nodes within the first 3 days after infection are the CD11c(+)CD8alpha(+)CD45RA(-) dendritic cells. Given that such class I-restricted presentation is essential for CTL priming, this implies that these conventional CD8alpha(+) dendritic cells are the key subset involved in CTL immunity to this virus.     

Accumulation of herpes simplex virus type 1 early and leaky-late proteins correlates with apoptosis prevention in infected human HEp-2 cells

We previously reported that a recombinant ICP27-null virus stimulated, but did not prevent, apoptosis in human HEp-2 cells during infection (M. Aubert and J. A. Blaho, J. Virol. 73:2803-2813, 1999). In the present study, we used a panel of 15 recombinant ICP27 mutant viruses to determine which features of herpes simplex virus type 1 (HSV-1) replication are required for the apoptosis-inhibitory activity. Each virus was defined experimentally as either apoptotic, partially apoptotic, or nonapoptotic based on infected HEp-2 cell morphologies, percentages of infected cells with condensed chromatin, and patterns of specific cellular death factor processing. Viruses d27-1, d1-5, d1-2, M11, M15, M16, n504R, n406R, n263R, and n59R are apoptotic or partially apoptotic in HEp-2 cells and severely defective for growth in Vero cells. Viruses d2-3, d3-4, d4-5, d5-6, and d6-7 are nonapoptotic, demonstrating that ICP27 contains a large amino-terminal region, including its RGG box RNA binding domain, which is not essential for apoptosis prevention. Accumulations of viral TK, VP16, and gD but not gC, ICP22, or ICP4 proteins correlated with prevention of apoptosis during the replication of these viruses. Of the nonapoptotic viruses, d4-5 did not produce gC, indicating that accumulation of true late gene products is not necessary for the prevention process. Analyses of viral DNA synthesis in HEp-2 cells indicated that apoptosis prevention by HSV-1 requires that the infection proceeds to the stage in which viral DNA replication takes place. Infections performed in the presence of the drug phosphonoacetic acid confirmed that the process of viral DNA synthesis and the accumulation of true late (gamma(2)) proteins are not required for apoptosis prevention. Based on our results, we conclude that the accumulation of HSV-1 early (beta) and leaky-late (gamma(1)) proteins correlates with the prevention of apoptosis in infected HEp-2 cells.     

Analysis of herpes simplex virus nucleoprotein complexes extracted from infected cells.

HEp-2 cells were infected with herpes simplex virus type 1 and labeled with [3H]thymidine and 14C-amino acids. Infected cells or nuclei prepared from them were extracted with Triton X-100 and NaCl, utilizing a method recently described, and the low-speed supernatant (extract) was partially purified by sedimentation on sucrose gradients. A nucleoprotein complex which sedimented as a wide peak around 200S was identified. The nucleoprotein complex contained viral DNA, which banded at the expected density in CsCl isopycnic gradients and was intact after measurements taken on electron microscopic photographic enlargements. The autoradiographic pattern of 14C-labeled proteins after electrophoresis showed that only a few of the virus-specific polypeptides were present in the nucleoprotein complexes, in particular, VP5, VP12, VP15.2, VP19, and VP24. Cellular histones were absent. The extracts and the nucleoprotein complexes were centrifuged to equilibrium in metrizamide density gradients without prefixation. Electron microscopic direct visualization of the nucleoprotein complexes after sucrose or metrizamide purification revealed that the proteins were preferentially associated with one end of the DNA molecule and formed large irregular terminal thickenings or capsid-like transparent shells enclosing polyglobular cores. No nucleosomes were observed on herpes simplex virus nucleoprotein complexes. The same type of complex was detected after phosphonoacetic acid addition, and grossly altered nucleocapsids were formed.     

Simultaneous tracking of capsid, tegument, and envelope protein localization in living cells infected with triply fluorescent herpes simplex virus 1

We report here the construction of a triply fluorescent-tagged herpes simplex virus 1 (HSV-1) expressing capsid protein VP26, tegument protein VP22, and envelope protein gB as fusion proteins with monomeric yellow, red, and cyan fluorescent proteins, respectively. The recombinant virus enabled us to monitor the dynamics of these capsid, tegument, and envelope proteins simultaneously in the same live HSV-1-infected cells and to visualize single extracellular virions with three different fluorescent emissions. In Vero cells infected by the triply fluorescent virus, multiple cytoplasmic compartments were found to be induced close to the basal surfaces of the infected cells (the adhesion surfaces of the infected cells on the solid growth substrate). Major capsid, tegument, and envelope proteins accumulated and colocalized in the compartments, as did marker proteins for the trans-Golgi network (TGN) which has been implicated to be the site of HSV-1 secondary envelopment. Moreover, formation of the compartments was correlated with the dynamic redistribution of the TGN proteins induced by HSV-1 infection. These results suggest that HSV-1 infection causes redistribution of TGN membranes to form multiple cytoplasmic compartments, possibly for optimal secondary envelopment. This is the first real evidence for the assembly of all three types of herpesvirus proteins-capsid, tegument, and envelope membrane proteins-in TGN.     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. III. Analysis of viral glycoproteins recognized by CTL clones by using recombinant herpes simplex viruses

Human cytotoxic T cell (CTL) clones specific for herpes simplex virus (HSV) type 1- and type 2-infected cells were generated and were analyzed with regard to the viral glycoproteins they recognize on autologous HSV-infected cells. By use of target cells infected with wild-type HSV strains, a gC deletion mutant of HSV-1, and HSV-1 X HSV-2 intertypic recombinants, some HSV-1-specific CTL clones were found to be directed against L region-encoded gA/B-1, and others against S region-encoded glycoproteins (gD-1 or gE-1). Some HSV-2-specific clones were found to be directed against L region-encoded gC-2, whereas others were directed against S region-encoded glycoproteins (gD-2, gE-2, or gG). These findings provide direct evidence that several HSV glycoproteins that are expressed on the surface of HSV-infected cells serve as recognition structures for human HSV-specific CTL.     

Differential in vitro activation of CD4+CD8- and CD8+CD4- herpes simplex virus-specific human cytotoxic T cells

In order to clarify the differential activation of CD4+ and CD8+ HSV-specific CTL, we compared the characteristics of CTL generated by different methods of in vitro HSV stimulation by treatment of effectors with anti-CD4 and anti-CD8 mAb and C after the elimination of nonspecific cytotoxic effector cells. Cell-free HSV mainly activated CD4+ CTL precursors, whereas HSV-infected fibroblasts were more effective in activating CD8+ CTL precursors than CD4+ CTL precursors. In addition, limiting dilution analyses with enriched T cells from two HSV-seropositive donors revealed that the frequency of HSV-specific CD4+ CTL precursors responsive to stimulation with free HSV was approximately 1/4,000 to 6,000 CD4+ T cells, whereas that of precursors responsive to stimulation with HSV-infected fibroblasts was approximately 1/19,000 to 22,000 CD4+ T cells. Conversely, the frequency of CD8+ CTL precursors in peripheral blood responsive to stimulation with free HSV was approximately 1/28,000 to 30,000 CD8+ T cells, whereas that of precursors responsive to stimulation with HSV-infected fibroblasts was approximately 1/10,000 to 11,000 CD8+ T cells. The present data suggest that generalized viral infection due to cell-free viruses is fought mainly by CD4+ CTL, which have previously been reported to possess both cytotoxicity and helper function, and that localized viral infection on HLA class II-negative fibroblasts is prevented from spreading to adjacent cells mainly by CD8+ CTL. Such differential activation of CD4+ and CD8+ CTL seems probable when considering the protective mechanisms against viral infection.     

Lifetimes of mRNA molecules directing the synthesis of viral proteins in herpes simplex virus-infected cells.

Possible variations in the functional lifetimes of herpes simplex virus type 1 mRNA molecules in infected HeLa cells were studied. As shown by the rate of decrease of radioactive amino acid incorporation into viral proteins after the addition of actinomycin, the average lifetime of early viral mRNA's are shorter than those for the late messenger species. In addition, when the viral proteins made after the addition of actinomycin were further analyzed by gel electrophoresis, it was found that messengers for individual viral proteins translated within the early or late time period also had some differences in their functional lifetimes. These results indicate that the synthesis of herpes simplex virus type 1 proteins during the replicative cycle is regulated in part by mechanisms controlling the functional lifetimes of viral mRNA's.     

Analysis of the interaction between the UL11 and UL16 tegument proteins of herpes simplex virus

The UL11 and UL16 tegument proteins of herpes simplex virus are conserved throughout the herpesvirus family. Previous studies have shown that these proteins interact, perhaps to link UL16-bound nucleocapsids to UL11, which resides on the cytoplasmic face of the trans-Golgi network, where maturation budding occurs. Little is known about the interaction except that it requires the leucine-isoleucine (LI) and acidic cluster motifs in UL11 and that no other viral proteins are involved. In particular, the important question of whether these two proteins bind to each other directly has not been addressed. Accordingly, UL11 and UL16 were expressed in bacteria, and the purified proteins were found to retain the ability to interact in a manner that was dependent upon the LI and acidic cluster. In an attempt to map the UL11-binding site contained in UL16, a large number of deletion mutants were constructed. The first 40 (nonconserved) amino acids were found to be dispensable, but all the other constructs failed to bind UL11 or had poor expression in transfected cells, suggesting that UL16 is very sensitive to alterations and probably lacks a multidomain structure. As an alternative strategy for identifying residues that are important for the interaction, the cysteines of UL16 were investigated, because many of these are highly conserved. Approximately half of the 20 cysteines in UL16 have been shown to be covalently modified by N-ethylmaleimide, and this treatment was found to block the interaction with UL11. Moreover, individual serine replacements of six of the most conserved cysteine residues were made, and four of these disrupted the interaction with UL11 without affecting protein stability. However, the UL11-UL16 interaction does not involve the formation of interspecies disulfide bonds, because binding occurred even when all the cysteines in UL11 were eliminated. Thus, UL16 directly interacts with UL11 and does so in a manner that requires free cysteines.     

Shared antigenic determinants between two distinct classes of proteins in cells infected with herpes simplex virus

Guinea pig antisera and mouse monoclonal antibodies against a 40,000-molecular-weight nucleocapsid protein (p40) of herpes simplex virus types 1 and 2 immunoprecipitated 40,000- and 80,000-molecular-weight classes of soluble proteins from infected cell extracts. The soluble 40,000-molecular-weight protein class (intracellular p40) appeared as a cluster of three to four closely spaced bands of proteins having molecular weights ranging between 39,000 and 45,000, whereas the soluble 80,000-molecular-weight protein class (intracellular p80) appeared as a doublet of bands. The peptide map of intracellular p40 closely resembled the maps of the p40 and p45 proteins of nucleocapsids, but it showed both differences and similarities when compared with the peptide map of intracellular p80. Pulse-chase experiments suggested that intracellular p80 was not a precursor of intracellular p40. We conclude that the intracellular p40 and p80 protein classes share common antigenic determinants, presumably reflecting similar amino acid sequences, although they have distinct differences in protein structure.