The structure of HLA-B8 complexed to an immunodominant viral determinant: peptide-induced conformational changes and a mode of MHC class I dimerization

EBV is a ubiquitous human pathogen that chronically infects up to 90% of the population. Persistent viral infection is characterized by latency and periods of viral replication that are kept in check by a strong antiviral CTL response. Despite the size of the EBV genome, CTL immunity focuses on only a few viral determinants but expands a large primary and memory response toward these epitopes. In unrelated HLA-B8(+) individuals, the response to the immunodominant latent Ag FLRGRAYGL from Epstein Barr nuclear Ag 3A is largely comprised of CTL clones with identical conserved alphabeta TCR structures. To better understand the structural correlates of Ag immunodominance and TCR selection bias, we have solved the crystal structure of the HLA-B8-FLRGRAYGL peptide complex to a resolution of 1.9 A. The structure confirms the importance of P3-Arg, P5-Arg, and P9-Leu as dominant anchor residues involved in peptide binding to HLA-B8. A bulged conformation of the bound peptide provides a structural basis for the critical role of the P7-Tyr residue in T cell recognition. The peptide also induces backbone and side-chain conformational changes in HLA-B8 that are transmitted along the peptide-binding groove in a domino effect. The HLA-B8-FLRGRAYGL complex crystallizes as a dimer in the asymmetric unit and is oriented such that both peptide ligands are projected in the same plane suggesting a higher order arrangement of MHC-peptide complexes that could be involved in formation of the class I Ag-loading complex or in T cell activation.     

Crystal structures of murine MHC Class I H-2 D(b) and K(b) molecules in complex with CTL epitopes from influenza A virus: implications for TCR repertoire selection and immunodominance

Cytotoxic T lymphocyte (CTL) responses against influenza A virus in C57BL/6 mice are dominated by a small number of viral peptides among many that are capable of binding to major histocompatibility complex (MHC) class I molecules. The basis of this limited immune recognition is unknown. Here, we present X-ray structures of MHC class I molecules in complex with two immunodominant epitopes (PA(224-233)/D(b) and PB1(703-711)/K(b)) and one non-immunogenic epitope (HA(468-477)/D(b)) of the influenza A virus. The immunodominant peptides are each characterized by a bulge at the C terminus, lifting P6 and P7 residues out of the MHC groove, presenting featured structural elements to T-cell receptors (TCRs). Immune recognition of PA(224-233)/D(b) will focus largely on the exposed P7 arginine residue. In contrast, the non-immunogenic HA(468-477) peptide lacks prominent features in this C-terminal bulge. In the K(b)-bound PB1(703-711) epitope, the bulge results from a non-canonical binding motif, such that the mode of presentation of this peptide strongly resembles that of D(b)-bound peptides. Given that PA(224-233)/D(b), PB1(703-711)/K(b) and the previously defined NP(366-374)/D(b) epitopes dominate the primary response to influenza A virus in C57BL/6 mice, our findings indicate that residues of the C-terminal bulge are important in selection of the immunodominant CTL repertoire.     

CD8(+) T cells from mice transnuclear for a TCR that recognizes a single H-2K(b)-restricted MHV68 epitope derived from gB-ORF8 help control infection

To study the CD8(+) T cell response against a mouse γ-herpes virus, we generated K(b)-MHV-68-ORF8(604-612)RAG(-/-) CD8(+) T cell receptor transnuclear (TN) mice as a source of virus-specific CD8(+) T cells. K(b)-ORF8-Tet(+) CD8(+) T cells, expanded in the course of a resolving MHV-68 infection, served as a source of nucleus donors. Various in vivo and ex vivo assay criteria demonstrated the fine specificity and functionality of TN cells. TN cells proliferated extensively in response to viral infection, helped control viral burden, and exhibited a phenotype similar to that of endogenous K(b)-ORF8-Tet(+) cells. When compared to OT-1 cells, TN cells displayed distinct properties in response to lymphopenia and cognate antigen stimulation, which may be attributable to the affinity of the TCR expressed by the TN cells. The availability of MHV-68-specific CD8(+) TCR TN mice provides a new tool for investigating aspects of host-pathogen interactions unique to γ-herpes viruses.     

Interaction between the DNA polymerase and single-stranded DNA-binding protein (infected cell protein 8) of herpes simplex virus 1

The herpes virus-encoded DNA replication protein, infected cell protein 8 (ICP8), binds specifically to single-stranded DNA with a stoichiometry of one ICP8 molecule/12 nucleotides. In the absence of single-stranded DNA, it assembles into long filamentous structures. Binding of ICP8 inhibits DNA synthesis by the herpes-induced DNA polymerase on singly primed single-stranded DNA circles. In contrast, ICP8 greatly stimulates replication of circular duplex DNA by the polymerase. Stimulation occurs only in the presence of a nuclear extract from herpes-infected cells. Appearance of the stimulatory activity in nuclear extracts coincides closely with the time of appearance of herpes-induced DNA replication proteins including ICP8 and DNA polymerase. A viral factor(s) may therefore be required to mediate ICP8 function in DNA replication.     

mRNA degradation by the virion host shutoff (Vhs) protein of herpes simplex virus: genetic and biochemical evidence that Vhs is a nuclease

During lytic infections, the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs. By accelerating the decay of all mRNAs, it helps redirect the cell from host to viral gene expression and facilitates the sequential expression of different classes of viral genes. While it is clear that Vhs induces mRNA degradation, it is uncertain whether it is itself an RNase or somehow activates a cellular enzyme. This question was addressed by using a combination of genetic and biochemical approaches. The Vhs homologues of alphaherpesviruses share sequence similarities with a family of mammalian, yeast, bacterial, and phage nucleases. To test the functional significance of these similarities, Vhs was mutated to alter residues corresponding to amino acids known to be critical to the nuclease activity of cellular homologues. In every instance, mutations that inactivated the nuclease activity of cellular homologues also abolished Vhs activity. Recent experiments showed that Vhs interacts with the cellular translation initiation factor eIF4H. In this study, the coexpression of Vhs and a glutathione S-transferase (GST)-eIF4H fusion protein in bacteria resulted in the formation of a complex of the proteins. The wild-type Vhs/GST-eIF4H complex was isolated and shown to have RNase activity. In contrast, Vhs mutations that altered key residues in the nuclease motif abolished the nuclease activity of the recombinant Vhs/GST-eIF4H complex. The results provide genetic and biochemical evidence that Vhs is an RNase, either alone or as a complex with eIF4H.     

Expression of herpes simplex virus type 1 glycoprotein L (gL) in transfected mammalian cells: evidence that gL is not independently anchored to cell membranes.

We expressed herpes simplex virus type 1 glycoprotein L (gL) in transfected cells to investigate whether it is independently anchored to plasma membranes or is membrane associated as a result of complex formation with gH. gL was detected by immunofluorescence microscopy at the surfaces of cotransfected cells when it was expressed with gH but not when it was expressed in the absence of gH or with a truncated form of gH, gHTrunc(792), which lacks the membrane-spanning region and terminates at amino acid 792. Immunoprecipitation studies of transfected-cell culture media revealed that gL was secreted from cells when expressed in the absence of gH and was secreted from cotransfected cells complexed with gHTrunc(792). These observations demonstrate that gL is not independently anchored to plasma membranes but is membrane associated as a result of complex formation with gH.     

The crystal structure of H-2D(b) complexed with a partial peptide epitope suggests a major histocompatibility complex class I assembly intermediate

In the absence of bound peptide ligands, major histocompatibility complex (MHC) class I molecules are unstable. In an attempt to determine the minimum requirement for peptide-dependent MHC class I stabilization, we have used short synthetic peptides derived from the Sendai virus nucleoprotein epitope (residues 324-332, 1FAPGNYPAL9) to promote its folding in vitro of H-2D(b). We found that H-2D(b) can be stabilized by the pentapeptide 5NYPAL9, which is equivalent to the C-terminal portion of the optimal nonapeptide and includes both the P5 and P9 anchor residues. We have crystallized the complex of the H-2D(b) molecule with the pentamer and determined the structure to show how a quasi-stable MHC class I molecule can be formed by occupancy of a single binding pocket in the peptide-binding groove.     

Inverted repeat regions of Marek''s disease virus DNA possess a structure similar to that of the a sequence of herpes simplex virus DNA and contain host cell telomere sequences.

The genomic structure of Marek's disease virus (MDV) is similar to those of the alphaherpesviruses herpes simplex virus (HSV) types 1 and 2. Sequence analysis of the junction region between the long component (L) and the short component (S) revealed the existence of an a-like sequence, similar in structure to the a sequence of HSV-1. Further study revealed that the MDV genome contains five copies of the a-like sequence within the long terminal repeat region as well as in the short terminal repeat region. The junction between the L and S components was found to contain 10 copies of the a-like sequence. Within the a-like sequence, a structure homologous to the DR2 of HSV was found to contain 17 copies of the telomeric sequence, GGGGTTA. There appears to be little to no sequence homology between the HSV a sequence and the MDV a-like sequence; however, the strong physical homology to its counterpart in HSV-1 suggests that the MDV a-like sequence may have the same functional homology (the domain for cleavage/packaging of the DNA into the viral capsids and for genomic inversion) as well.     

Rapid CD8+ T cell repertoire focusing and selection of high-affinity clones into memory following primary infection with a persistent human virus: human cytomegalovirus

To investigate the mechanism of selection of individual human CD8+ T cell clones into long-term memory following primary infection with a persistent human virus (human CMV (HCMV)), we undertook a longitudinal analysis of the diversity of T cell clones directed toward an immunodominant viral epitope: we followed this longitudinally from early T cell expansion through the contraction phase and selection into the memory pool. We show that following initial HCMV infection, the early primary response against a defined epitope was composed of diverse clones possessing many different TCR Vbeta segments. Longitudinal analysis showed that this usage rapidly focused predominantly on a single TCR Vbeta segment within which dominant clones frequently had public TCR usage, in contrast to subdominant or contracted clones. Longitudinal clonotypic analysis showed evidence of disproportionate contraction of certain clones that were abundant in the primary response, and late expansion of clones that were subdominant in the primary response. All dominant clones selected into memory showed similar high functional avidity of their TCR, whereas two clones that greatly contracted showed substantially lower avidity. Expression of the IL-7R is required for survival of murine effector CD8+ T cells into memory, but in primary HCMV infection IL-7R was not detected on circulating Ag-specific cells until memory had been established. Thus, the oligoclonal T cell repertoire against an immunodominant persistent viral epitope is established early in primary infection by the rapid selection of public clonotypes, rather than being a stochastic process.     

Protection of mice against herpes simplex virus infection by a Lactobacillus casei preparation (LC 9018) in combination with inactivated viral antigen

Heat-killed Lactobacillus casei YIT 9018 (LC 9018) cells enhanced the resistance to herpes simplex virus type 1 (HSV-1) in adult mice, but not significantly. The protection of mice against HSV-1 infection and the production of neutralizing antibodies were significantly enhanced by the administration of LC 9018 in combination with inactivated HSV-1 antigen. The optimal enhancement of resistance was seen in mice 14 days after the simultaneous administration of these substances. The resistance to HSV-1 infection in mice could be transferred with peritoneal exudate cells from syngeneic mice previously treated with LC 9018 alone and LC 9018 in combination with inactivated HSV-1 antigen or with thioglycollate broth, whereas the transfer of peritoneal exudate cells induced by thioglycollate broth alone and of spleen cells induced by LC 9018 in combination with thioglycollate broth or by thioglycollate broth alone was not effective. These results suggest that mouse peritoneal macrophages induced by the administration of LC 9018 in combination with inactivated HSV-1 antigen may play an important role in host defense mechanisms against HSV-1 infection.     

Generation and phenotype of cell lines derived from CF and non-CF mice that carry the H-2K(b)-tsA58 transgene

Tracheal, renal, salivary, and pancreatic epithelial cells from cystic fibrosis [CF; cystic fibrosis transmembrane conductance regulator (CFTR) -/-] and non-CF mice that carry a temperature-sensitive SV40 large T antigen oncogene (ImmortoMouse) were isolated and maintained in culture under permissive conditions (33 degrees C with interferon-gamma). The resultant cell lines have been in culture for >1 year and 50 passages. Each of the eight cell lines form polarized epithelial barriers and exhibit regulated, electrogenic ion transport. The four non-CF cell lines (mTEC1, mCT1, mSEC1, and mPEC1) express cAMP-regulated Cl(-) permeability and cAMP-stimulated Cl(-) secretion. In contrast, the four CFTR -/- cell lines (mTEC1-CF, mCT1-CF, mSEC1-CF, and mPEC1-CF) each lack cAMP-stimulated Cl(-) secretory responses. Ca(2+)-activated Cl(-) secretion is retained in both CF and non-CF cell lines. Thus we have generated genetically well-matched epithelial cell lines from several tissues relevant to cystic fibrosis that either completely lack CFTR or express endogenous levels of CFTR. These cell lines should prove useful for studies of regulation of epithelial cell function and the role of CFTR in cell physiology.     

Identification of a replication-defective herpes simplex virus for recombinant adeno-associated virus type 2 (rAAV2) particle assembly using stable producer cell lines

BACKGROUND: The development of stable producer cell lines for recombinant adeno-associated virus (rAAV) assembly is a strategy followed by many groups to develop scalable production methods suitable for good manufacturing practice (GMP) requirements. The major drawback of this method lies in the requirement for replicating adenovirus (Ad) for rAAV assembly. In the present study, we analyzed the ability of several replication-defective herpes simplex type 1 (HSV-1) helper viruses to induce rAAV2 particle production from stable producer cell lines. METHODS: Several stable rAAV producer cell clones were infected with wild-type and replication-defective HSV strains and analyzed for rep-cap gene amplification, viral protein synthesis and rAAV titers achieved. In vivo analysis following rAAV injection in the murine brain was also conducted to evaluate the toxicity and biopotency of the rAAV stocks. RESULTS: We demonstrated that an HSV strain mutated in the UL30 polymerase gene could efficiently be used in this context, resulting in rAAV titers similar to those measured with wild-type HSV or Ad. Importantly, with respect to clinical developments, the use of this mutant resulted in rAAV stocks which were consistently devoid of contaminating HSV particles and fully active in vivo in the murine central nervous system with no detectable toxicity. CONCLUSIONS: This study, together with our previous report describing a rAAV chromatography-based purification process, contributes to the definition of an entirely scalable process for the generation of rAAV particles.     

Identification of a divalent metal cation binding site in herpes simplex virus 1 (HSV-1) ICP8 required for HSV replication

Herpes simplex virus 1 (HSV-1) ICP8 is a single-stranded DNA-binding protein that is necessary for viral DNA replication and exhibits recombinase activity in vitro. Alignment of the HSV-1 ICP8 amino acid sequence with ICP8 homologs from other herpesviruses revealed conserved aspartic acid (D) and glutamic acid (E) residues. Amino acid residue D1087 was conserved in every ICP8 homolog analyzed, indicating that it is likely critical for ICP8 function. We took a genetic approach to investigate the functions of the conserved ICP8 D and E residues in HSV-1 replication. The E1086A D1087A mutant form of ICP8 failed to support the replication of an ICP8 mutant virus in a complementation assay. E1086A D1087A mutant ICP8 bound DNA, albeit with reduced affinity, demonstrating that the protein is not globally misfolded. This mutant form of ICP8 was also recognized by a conformation-specific antibody, further indicating that its overall structure was intact. A recombinant virus expressing E1086A D1087A mutant ICP8 was defective in viral replication, viral DNA synthesis, and late gene expression in Vero cells. A class of enzymes called DDE recombinases utilize conserved D and E residues to coordinate divalent metal cations in their active sites. We investigated whether the conserved D and E residues in ICP8 were also required for binding metal cations and found that the E1086A D1087A mutant form of ICP8 exhibited altered divalent metal binding in an in vitro iron-induced cleavage assay. These results identify a novel divalent metal cation-binding site in ICP8 that is required for ICP8 functions during viral replication.     

Characterization of a BHK(TK-) cell clone resistant to postattachment entry by herpes simplex virus types 1 and 2.

BHK(TK-) cells selected for resistance to polyethylene glycol-mediated fusion give rise to clones that are resistant to herpes simplex virus (HSV) infection. We have characterized one such clone, designated 95-19, and found that it is resistant to entry of HSV type 1 (HSV-1), HSV-2, and the related alphaherpesvirus pseudorabies virus (PRV). Single-step growth experiments show no detectable replication of multiple strains of HSV-1 and HSV-2 on 95-19 cells. Three lines of evidence suggest that these cells are resistant to postattachment entry. (i) Measurements of binding of radiolabeled virus show that heparin-sensitive binding of HSV-1 and HSV-2 to 95-19 cells is identical to binding to BHK(TK-) cells, suggesting that the block to replication occurs after attachment to heparan sulfate proteoglycan. (ii) 95-19 cells exposed to HSV-1 or HSV-2 at high multiplicity show no detectable immediate-early (IE) mRNA expression. (iii) Exposure of attached virus and cells to polyethylene glycol results in partial recovery of both IE gene expression and virus yield in single-step growth. The degrees of recovery of single-step yield and IE gene expression are similar, suggesting that the only block to single-step replication is at the point of virus entry and that these cells are deficient in some cellular factor required for efficient postattachment entry of free virus. 95-19 cells are also highly resistant to entry by cell-to-cell spread, suggesting that the same cellular factor participates in both types of entry.     

Development of a glycoprotein D-expressing dominant-negative and replication-defective herpes simplex virus 2 (HSV-2) recombinant viral vaccine against HSV-2 infection in mice

Using the T-REx (Invitrogen, California) gene switch technology and a dominant-negative mutant polypeptide of herpes simplex virus 1 (HSV-1)-origin binding protein UL9, we previously constructed a glycoprotein D-expressing replication-defective and dominant-negative HSV-1 recombinant viral vaccine, CJ9-gD, for protection against HSV infection and disease. It was demonstrated that CJ9-gD is avirulent following intracerebral inoculation in mice, cannot establish detectable latent infection following different routes of infection, and offers highly effective protective immunity against primary HSV-1 and HSV-2 infection and disease in mouse and guinea pig models of HSV infections. Given these favorable safety and immunological profiles of CJ9-gD, aiming to maximize levels of HSV-2 glycoprotein D (gD2) expression, we have constructed an ICP0 null mutant-based dominant-negative and replication-defective HSV-2 recombinant, CJ2-gD2, that contains 2 copies of the gD2 gene driven by the tetracycline operator (tetO)-bearing HSV-1 major immediate-early ICP4 promoter. CJ2-gD2 expresses gD2 as efficiently as wild-type HSV-2 infection and can lead to a 150-fold reduction in wild-type HSV-2 viral replication in cells coinfected with CJ2-gD2 and wild-type HSV-2 at the same multiplicity of infection. CJ2-gD2 is avirulent following intracerebral injection and cannot establish a detectable latent infection following subcutaneous (s.c.) immunization. CJ2-gD2 is a more effective vaccine than HSV-1 CJ9-gD and a non-gD2-expressing dominant-negative and replication-defective HSV-2 recombinant in protection against wild-type HSV-2 genital disease. Using recall response, we showed that immunization with CJ2-gD2 elicited strong HSV-2-specific memory CD4(+) and CD8(+) T-cell responses. Collectively, given the demonstrated preclinical immunogenicity and its unique safety profiles, CJ2-gD2 represents a new class of HSV-2 replication-defective recombinant viral vaccines in protection against HSV-2 genital infection and disease.     

Murine interstitial nephritis. III. The selection of phenotypic (Lyt and L3T4) and idiotypic (RE-Id) T cell preferences by genes in Igh-1 and H-2K characterizes the cell-mediated potential for disease expression: susceptible mice provide a unique effector T cell repertoire in response to tubular antigen

The effector T cell repertoire in experimental interstitial nephritis was examined in a variety of susceptible and nonsusceptible mice. We observed that L3T4+ effector T cells in disease-susceptible mice disappear soon after immunization in preference to the emergence of Lyt-2+ effector cells. These latter cells respond with delayed-type hypersensitivity to tubular antigen in the context of H-2K. Such cells also express idiotypes (RE-Id) shared with kidney-bound alpha TBM-Ab that are regulated by an interactional effect of genes in Igh-1 and H-2K. These Lyt-2+ effector cells can be removed from renal infiltrates, and the transfer of similar cells under the renal capsule of naive mice results, within 5 days, in local interstitial nephritis. Nonsusceptible mice, however, not having these immune response genes, produce either L3T4+, Lyt-1+, RE-Id- effector T cells, which only respond to tubular antigen in the context of I-A, or Lyt-2+, RE-Id- T cells, which may lack very fine specificity. These findings suggest that susceptible mice carry a unique set of immune response genes that promote a T cell selection process that operates after induction, during the differentiation and development of disease-producing effector T cells.     

CD11b (Mac-1): a marker for CD8+ cytotoxic T cell activation and memory in virus infection.

We have found that CD11b, a cell surface integrin of macrophages, granulocytes, and NK cells, is expressed by a subset of CD8+ T cells that include both the active virus-specific CTL and the virus-specific memory CTL populations. CD8+CD11b+ cells comprise less than 3% of naive mouse splenocytes, but after lymphocytic choriomeningitis virus (LCMV) infection increase by 9- to 12-fold by the peak (day 8) of the virus-specific CTL response. Depletion of day-8 splenocytes with anti-Mac-1 and C' or enrichment by sorting for CD11b+ or CD8+CD11b+ spleen cells demonstrated that LCMV-specific CTL are CD11b+. The CD11b+ subpopulation also contained the bulk of the IL-2-responsive CD8+ cells. MEL-14, a homing marker down-regulated on activated T cells, was down-regulated on the majority of CD8+ cells that became CD11b+. Less than 1% of LCMV-immune splenic lymphocytes expressed CD11b. Antibody and C' depletion of this population severely impaired the ability of immune splenocytes to respond to in vitro secondary stimulation with LCMV-infected peritoneal macrophages, but did not affect the generation of a primary allospecific CTL response in MLC. Mixing of CD8-depleted and CD11b-depleted LCMV-immune splenocytes failed to restore the ability of these cells to mount a virus-specific memory CTL response, indicating that a cell coexpressing CD8 and CD11b is essential for this response. As determined by limiting dilution analysis, the precursors for the LCMV-specific memory CTL response were enriched in the CD11b+ population of LCMV-immune splenocytes. CD11b stained far fewer CD8+ splenocytes from naive mice than did CD44 (Pgp-1), and among immune splenocytes it identified a small subpopulation of CD44hi cells, indicating that CD11b may be the best single marker available for discriminating between naive and memory CD8+ T cells.     

Deletion of the virion host shutoff protein (vhs) from herpes simplex virus (HSV) relieves the viral block to dendritic cell activation: potential of vhs- HSV vectors for dendritic cell-mediated immunotherapy

Herpes simplex virus (HSV) infects dendritic cells (DC) efficiently but with minimal replication. HSV, therefore, appears to have evolved the ability to enter DC even though they are nonpermissive for virus growth. This provides a potential utility for HSV in delivering genes to DC for vaccination purposes and also suggests that the life cycle of HSV usually includes the infection of DC. However, DC infected with HSV usually lose the ability to become activated following infection (M. Salio, M. Cella, M. Suter, and A. Lanzavecchia, Eur. J. Immunol. 29:3245-3253, 1999; M. Kruse, O. Rosorius, F. Kratzer, G. Stelz, C. Kuhnt, G. Schuler, J. Hauber, and A. Steinkasserer, J. Virol. 74:7127-7136, 2000). We report that for DC to retain the ability to become activated following HSV infection, the virion host shutoff protein (vhs) must be deleted. vhs usually functions to destabilize mRNA in favor of the production of HSV proteins in permissive cells. We have found that it also plays a key role in the inactivation of DC and is therefore likely to be important for immune evasion by the virus. Here, vhs would be anticipated to prevent DC activation in the early stages of infection of an individual with HSV, reducing the induction of cellular immune responses and thus preventing virus clearance during repeated cycles of virus latency and reactivation. Based on this information, replication-incompetent HSV vectors with vhs deleted which allow activation of DC and the induction of specific T-cell responses to delivered antigens have been constructed. These responses are greater than if DC are loaded with antigen by incubation with recombinant protein.     

Immunization with a replication-deficient mutant of herpes simplex virus type 1 (HSV-1) induces a CD8+ cytotoxic T-lymphocyte response and confers a level of protection comparable to that of wild-type HSV-1.

Replication-deficient viruses provide an attractive alternative to conventional approaches used in the induction of antiviral immunity. We have quantitatively evaluated both the primary and memory cytotoxic T-lymphocyte (CTL) responses elicited by immunization with a replication-deficient mutant of herpes simplex virus type 1 (HSV-1). In addition, we have examined the potential role of these CTL in protection against HSV infection. Using bulk culture analysis and limiting-dilution analysis, we have shown that a replication-deficient virus, d301, generates a strong primary CTL response that is comparable to the response induced by the wild type-strain, KOS1.1. Furthermore, the CTL induced by d301 immunization recognized the immunodominant, H-2Kb-restricted, CTL recognition epitope gB498-505 to a level similar to that for CTL from KOS1.1-immunized mice. The memory CTL response evoked by d301 was strong and persistent, even though the frequencies of CTL were slightly lower than the frequencies of CTL induced by KOS1.1. Adoptive transfer studies indicated that both the CD8+ and the CD4+ T-cell responses generated by immunization with d301 and KOS1.1 were able to limit the extent of a cutaneous HSV infection to comparable levels. Overall, these results indicate that viral replication is not necessary to elicit a potent and durable HSV-specific immune response and suggest that replication-deficient viruses may be effective in eliciting protection against viral pathogens.