CD154 is essential for protective immunity in experimental salmonella infection: evidence for a dual role in innate and adaptive immune responses

CD40-CD154 interactions are of central importance in the induction of humoral and cellular immune responses. In the present study, CD154-deficient (CD154-/-) mice were used to assess the role of CD40-CD154 interactions in regulating the immune response to a systemic Salmonella infection. Compared with C57BL/6 (CD154+/+) controls, CD154-/- mice were hypersusceptible to infection by an attenuated strain of Salmonella enterica serovar Typhimurium (S. typhimurium), as evidenced by decreased survival rate and mean time to death, which correlated with increased bacterial burden and persistence in target organs. CD154-/- mice exhibited a defect both in the production of IL-12, IFN-gamma, and NO during the acute phase of the disease and in the generation of Salmonella-specific Ab responses and Ig isotype switching. Furthermore, when CD154-/- animals were administered a sublethal dose of attenuated S. typhimurium and subsequently challenged with a virulent homologous strain, all mice succumbed to an overwhelming infection. Similar treatment of CD154+/+ mice consistently resulted in > or =90% protection. The lack of protective immunity in CD154-/- mice correlated with a decreased T cell recall response to Salmonella Ags. Significant protection against virulent challenge was conferred to presensitized CD154-/- mice by transfer of serum or T cells from immunized CD154+/+ mice. For best protection, however, a combination of immune serum and T cells was required. We conclude that intercellular communications via the CD40-CD154 pathway play a critical role in the induction of type 1 cytokine responses, memory T cell generation, Ab formation, and protection against primary as well as secondary Salmonella infections.     

Cutting edge: C3d functions as a molecular adjuvant in the absence of CD21/35 expression

Complement component C3 covalently attaches to Ags following activation, where the C3d cleavage fragment can function as a molecular adjuvant to augment humoral immune responses. C3d is proposed to exert its adjuvant-like activities by targeting Ags to the C3d receptor (CD21/35) expressed by B cells and follicular dendritic cells. To directly assess the importance of CD21/35 in mediating the immunostimulatory effects of C3d, CD21/35-deficient (CD21/35(-/-)) mice were immunized with streptavidin (SA), SA-C3dg tetramers, recombinant HIV gp120 (gp120), or gp120 fused with linear multimers of C3d. Remarkably, SA- and gp120-specific Ab responses were significantly augmented in CD21/35(-/-) mice when these Ags were complexed with C3d in comparison to Ag alone. In fact, primary and secondary Ab responses and Ab-forming cell responses of CD21/35(-/-) mice approached those of wild-type mice immunized with SA-C3dg and gp120-C3d. Thus, C3d can function as a molecular adjuvant in the absence of CD21/35 expression.     

Selective requirement for CD40-CD154 in drug-induced type 1 versus type 2 responses to trinitrophenyl-ovalbumin

CD154 is transiently expressed by activated T cells and interacts with CD40 on B cells, dendritic cells, macrophages, and monocytes. This costimulatory receptor-ligand couple seems decisive in Ag-driven immune responses but may be differentially involved in type 1 vs type 2 responses. We studied the importance of CD40-CD154 in both responses using the reporter Ag popliteal lymph node assay in which selectively acting drugs generate clearly polarized type 1 (streptozotocin) or type 2 (D-penicillamine, diphenylhydantoin) responses to a constant coinjected Ag in the same mouse strain. Treatment of mice with anti-CD154 reduced characteristic immunological parameters in type 2 responses (B and CD4(+) T cell proliferation, IgG1 and IgE Abs, and IL-4 secretion) and only slightly affected the type 1 response (small decrease in IFN-gamma production, influx of CD11c(+) and F4/80(+) cells, and prevention of architectural disruption of the lymph node, but no effect on IgG2a Ab and TNF-alpha secretion or B and CD4(+) T cell proliferation). The findings indicate that the CD40-CD154 costimulatory interaction is a prerequisite in drug-induced type 2 responses and is only marginally involved in type 1 responses. The observed expression patterns of CD80 and CD86 on different APC (B cells in type 2 and dendritic cells in type 1) may be responsible for this discrepancy.     

Restoration of the antibody response to IgE/antigen complexes in CD23-deficient mice by CD23+ spleen or bone marrow cells

Mice immunized with IgE/Ag complexes produce significantly more Ag-specific Abs than mice immunized with Ag alone. The enhancement is mediated via the low-affinity receptor for IgE (FcepsilonRII or CD23), as shown by its complete absence in mice pretreated with mAbs specific for CD23 and in CD23-deficient mice. Because the constitutive expression of murine CD23 is limited to B cells and follicular dendritic cells (FDCs), one of these cell types is likely to be involved. One of the suggested modes of action of IgE/CD23 is to increase the ability of B cells to present Ag to T cells, as demonstrated to take place in vitro. Another possibility is that FDCs capture the IgE/Ag complexes and present these directly to B cells. The purpose of the present study was to determine whether CD23+ B cells or FDCs are responsible for the IgE/CD23-mediated enhancement of specific Ab responses in vivo. We show that the enhancement is completely restored in irradiated CD23-deficient mice reconstituted with CD23+ spleen or bone marrow cells. In these mice, the B cells are CD23+ and the FDCs are presumably CD23- because the FDCs are radiation resistant and are reported not to be replaced by donor cells after this type of cell transfer. In contrast, enhancement was not restored in irradiated wild-type mice reconstituted with CD23- cells. These results indicate that CD23+ B cells, and not FDCs, are the cells that capture IgE/Ag complexes and induce enhancement of Ab responses in vivo.     

Bovine herpesvirus 1 VP22 enhances the efficacy of a DNA vaccine in cattle

For this study, the intercellular trafficking ability of bovine herpesvirus 1 (BHV-1) VP22 was applied to improve the efficacy of a DNA vaccine in calves. A plasmid encoding a truncated version of glycoprotein D (tgD) fused to VP22 was constructed. The plasmid encoding tgD-VP22 elicited significantly enhanced and more balanced immune responses than those induced by a plasmid encoding tgD. Furthermore, protection against a BHV-1 challenge was obtained in calves immunized with the plasmid encoding tgD-VP22, as shown by significant reductions in viral excretion. However, less significant protection was observed for animals vaccinated with the tgD-expressing plasmid, correlating with the lower level of immunity observed prechallenge. This is the first report of the use of VP22 as a transport molecule in the context of a DNA vaccine for a large animal species.     

Oral DNA vaccination in utero induces mucosal immunity and immune memory in the neonate

Infectious diseases are responsible for a significant number of deaths during the first weeks of life. Some of the salient pathogens include HSV, HIV, hepatitis B virus, group B streptococcus, Haemophilus sp., and Chlamydia sp. The vertical transmission of many of these pathogens significantly increases the risk of neonatal infection. We recently reported that oral DNA immunization in utero induced high serum Ab titers and cell-mediated immunity in fetal lambs. In this study, we demonstrate immune memory and mucosal immunity in newborn lambs following oral DNA immunization of the fetus. A single oral exposure in utero to plasmid DNA encoding a truncated form of glycoprotein D of bovine herpesvirus-1 induced detectable immune responses in 80% (12 of 15) of newborn lambs. There was no evidence for the induction of immune tolerance in nonresponding lambs. Responding lambs displayed both systemic and mucosal immune responses and reduced virus shedding following intranasal challenge. Furthermore, strong anamnestic responses were evident for at least 3 mo after birth. The efficacy of in utero oral DNA immunization was further demonstrated with the hepatitis B surface Ag, and protective serum Ab titers occurred in 75% of immunized lambs. Thus, the present investigation confirms that oral DNA immunization in utero can induce both mucosal and systemic immune responses in the neonate and that this immunity has the potential to prevent vertical disease transmission.     

Passively acquired antibodies suppress humoral but not cell-mediated immunity in mice immunized with live attenuated respiratory syncytial virus vaccines

A respiratory syncytial virus (RSV) vaccine will need to be administered by 1 mo of age to protect young infants; therefore, it will need to be effective in the presence of maternally acquired RSV Abs. In the present study, the immunogenicity and efficacy of two live attenuated RSV vaccine candidates of different level of attenuation were evaluated in mice passively immunized with varying quantities of RSV Abs. The replication of the RSV vaccines was suppressed in the lower, but not the upper, respiratory tract of the passively immunized mice. Immunization with either vaccine candidate was highly efficacious against challenge with wild-type RSV in both passively immunized and control mice. Nonetheless, a high level of immunity was seen even in passively/actively immunized animals that failed to develop a humoral immune response, suggesting that T cells mediated the immunity. Depletion of CD4+ and CD8+ T cells in passively/actively immunized and control animals at the time of challenge with wild-type RSV demonstrated that CD4+ and CD8+ T cells made significant independent contributions to the restriction of replication of RSV challenge virus in both the upper and lower respiratory tracts. Although passively acquired serum RSV Abs suppressed the primary systemic and mucosal Ab responses of IgM, IgG, and IgA isotypes, B lymphocytes were nevertheless primed for robust secondary Ab responses. Thus, immunity mediated by CD4+ and CD8+ T cells and Abs can be readily induced in mice by live RSV vaccine candidates in the presence of physiologic levels of RSV neutralizing Abs.     

Follicular helper NKT cells induce limited B cell responses and germinal center formation in the absence of CD4(+) T cell help

B cells require MHC class II (MHC II)-restricted cognate help and CD40 engagement by CD4(+) T follicular helper (T(FH)) cells to form germinal centers and long-lasting Ab responses. Invariant NKT (iNKT) cells are innate-like lymphocytes that jumpstart the adaptive immune response when activated by the CD1d-restricted lipid α-galactosylceramide (αGalCer). We previously observed that immunization of mice lacking CD4(+) T cells (MHC II(-/-)) elicits specific IgG responses only when protein Ags are mixed with αGalCer. In this study, we investigated the mechanisms underpinning this observation. We find that induction of Ag-specific Ab responses in MHC II(-/-) mice upon immunization with protein Ags mixed with αGalCer requires CD1d expression and CD40 engagement on B cells, suggesting that iNKT cells provide CD1d-restricted cognate help for B cells. Remarkably, splenic iNKT cells from immunized MHC II(-/-) mice display a typical CXCR5(hi)programmed death-1(hi)ICOS(hi)Bcl-6(hi) T(FH) phenotype and induce germinal centers. The specific IgG response induced in MHC II(-/-) mice has shorter duration than that developing in CD4-competent animals, suggesting that iNKT(FH) cells preferentially induce transient rather than long-lived Ab responses. Together, these results suggest that iNKT cells can be co-opted into the follicular helper function, yet iNKT(FH) and CD4(+) T(FH) cells display distinct helper features, consistent with the notion that these two cell subsets play nonredundant functions throughout immune responses.     

CD36 is differentially expressed on B cell subsets during development and in responses to antigen

Of a number of mAbs made by immunization with sort-purified marginal zone (MZ) B cells, one was shown to recognize the mouse scavenger receptor CD36. Although CD36 is expressed by most resting MZ B cells and not by follicular and B1 B cells, it is rapidly induced on follicular B cells in vitro following TLR and CD40 stimulation. In response to T-independent and T-dependent Ag challenge, we found that CD36 was expressed on IgM+ plasma cells, but down-regulated on isotype-switched plasma cells in vivo. Although development, localization, and phenotype of MZ B cells in CD36-/- mice appeared normal, there was a minor block in the transitional stages of mature B cell development. In both primary and secondary Ab responses to heat-killed Streptococcus pneumoniae (R36A strain), both phosphoryl choline (PC)-specific IgM and IgG levels in CD36-/- mice were slightly reduced compared with wild-type mice. In addition, mice deficient in both TLR2 and CD36 produced significantly reduced levels of anti-PC IgG titers than those of single gene-deficient mice, suggesting that they may cooperate in an anti-PC Ab response. Collectively, these results show that CD36 does not affect the development of B cells, but modulates both primary and secondary anti-PC Ab responses during S. pneumoniae infection similarly to TLR2.     

The identification and characterization of a ligand for bovine CD5

CD5, a type I glycoprotein expressed by T cells and a subset of B cells, is thought to play a significant role in modulating Ag receptor signaling. Previously, our laboratory has shown that bovine B cells are induced to express this key regulatory molecule upon Ag receptor cross-linking. To date, a ligand has not been described for bovine CD5. Given the importance ligand binding presumably plays in the functioning of CD5 on this B cell subset and on T cells, we sought to characterize the ligand for this protein using a bovine CD5-human IgG1 (CD5Ig) fusion protein produced by both mammalian and yeast cells. As determined by CD5Ig binding, expression of this ligand is negative to low on freshly isolated lymphocytes, with low-density expression being limited to activated B cells. Activation with LPS, PMA, and calcium ionophore, or ligation of CD40 alone or in combination with anti-IgM, resulted in B cell-specific expression of this ligand. Interestingly, activation through B cell Ag receptor cross-linking alone, although able to induce CD5 expression, did not result in expression of CD5 ligand (CD5L). In addition, we demonstrate a functional role for CD5L as a costimulatory molecule that augments CD40L-stimulated B cell proliferation. Finally, immunoprecipitation with CD5Ig suggests that the ligand characterized in this study has a molecular mass of approximately 200 kDa. The data reported herein, as well as future studies aimed at further characterizing this newly identified bovine CD5L, will undoubtedly aid in understanding the role that the CD5-CD5L interaction plays in immune responses.     

Basic mechanisms of DNA-raised antibody responses to intramuscular and gene gun immunizations

DNA-raised antibody (Ab) responses have been compared for the dependence on CD4+ and CD8+ cells, the longevity of functional antigen (Ag) expression, and the nature of the Ag-presenting cell after intramuscular (i.m.) and gene gun inoculations. A plasmid expressing the hemagglutinin (HA) glycoprotein of influenza virus was used for immunizations of BALB/c mice. Intramuscular and gene gun-raised Abs had similar dependencies on CD4+ and CD8+ cells but different temporal patterns of functional Ag expression. The two methods of DNA immunization also appeared to have different frequencies or types of Ag-presenting cells in the draining lymph nodes and spleen. For both methods of DNA delivery, Ab was independent of CD8+ cells but dependent on CD4+ cells. The CD4 dependence occurred at priming but not booster immunizations and resulted in a 1-month delay in the Ab response. Temporal T-cell transfers from TCR+/+ mice into immunized TCR-/- mice revealed the persistence of DNA-expressed Ag for up to 1 month after both i.m. and gene gun inoculations. For gene gun, but not i.m. immunizations, approximately 90% of the functional Ag expression was lost by 1 week, consistent with the sloughing of the epidermal target site. Despite similar titers of raised Ab, Ag-presenting dendritic cells could be detected in the draining lymph nodes and spleen of gene gun- but not i.m. DNA-immunized mice. In the gene gun-immunized mice, Ag-presenting dendritic cells appeared in the draining lymph nodes before the spleen.     

Humoral immune responses in Cr2-/- mice: enhanced affinity maturation but impaired antibody persistence

Deficiency in CD21/CD35 by disruption of the Cr2 loci leads to impaired humoral immune responses. In this study, we detail the role of CD21/CD35 on Ab responses to the hapten (4-hydroxy-3-nitrophenyl)acetyl conjugated to chicken gamma-globulin. Surprisingly, Cr2-/- mice generate significant Ab responses and germinal center (GC) reactions to low doses of this Ag in alum, although the magnitude of their responses is much reduced in comparison with those of Cr2+/- and C57BL/6 controls. Increasing Ag dose partially corrected this deficit. In situ study of the somatic genetics of GC B cells demonstrated that VDJ hypermutation does not require CD21/CD35, and Cr2-/- mice exhibited enhanced affinity maturation of serum Ab in the post-GC phase of the primary response. On the other hand, Cr2-/- mice displayed accelerated loss of serum Ab and long-lived Ab-forming cells. These observations suggest that B cell activation/survival signals mediated by CD21 and/or the retention of Ag by CD21/CD35 play important roles in the generation, quality, and maintenance of serum Ab.     

CD40, but not CD154, expression on B cells is necessary for optimal primary B cell responses

CD40 is an important costimulatory molecule for B cells as well as dendritic cells, monocytes, and other APCs. The ligand for CD40, CD154, is expressed on activated T cells, NK cells, mast cells, basophils, and even activated B cells. Although both CD40(-/-) and CD154(-/-) mice have impaired ability to isotype switch, form germinal centers, make memory B cells, and produce Ab, it is not entirely clear whether these defects are intrinsic to B cells, to other APCs, or to T cells. Using bone marrow chimeric mice, we investigated whether CD40 or CD154 must be expressed on B cells for optimal B cell responses in vivo. We demonstrate that CD40 expression on B cells is required for the generation of germinal centers, isotype switching, and sustained Ab production, even when other APCs express CD40. In contrast, the expression of CD154 on B cells is not required for the generation of germinal centers, isotype switching, or sustained Ab production. In fact, B cell responses are completely normal when CD154 expression is limited exclusively to Ag-specific T cells. These results suggest that the interaction of CD154 expressed by activated CD4 T cells with CD40 expressed by B cells is the primary pathway necessary to achieve B cell activation and differentiation and that CD154 expression on B cells does not noticeably facilitate B cell activation and differentiation.     

CD22 regulates time course of both B cell division and antibody response

Because pathogens induce infectious symptoms in a time-dependent manner, a rapid immune response is beneficial for defending hosts from pathogens, especially those inducing acute infectious diseases. However, it is largely unknown how the time course of immune responses is regulated. In this study, we demonstrate that B cells deficient in the inhibitory coreceptor CD22 undergo accelerated cell division after Ag stimulation, resulting in rapid generation of plasma cells and Ab production. This finding indicates that CD22 regulates the time course of B cell responses and suggests that CD22 is a good target to shorten the time required for Ab production, thereby augmenting host defense against acute infectious diseases as "universal vaccination."     

Tumor immunotherapy by epicutaneous immunization requires langerhans cells

A role for Langerhans cells (LC) in the induction of immune responses in the skin has yet to be conclusively demonstrated. We used skin immunization with OVA protein to induce immune responses against OVA-expressing melanoma cells. Mice injected with OVA-specific CD8(+) T cells and immunized with OVA onto barrier-disrupted skin had increased numbers of CD8(+) T cells in the blood that produced IFN-gamma and killed target cells. These mice generated accelerated cytotoxic responses after secondary immunization with OVA. Prophylactic or therapeutic immunization with OVA onto barrier-disrupted skin inhibited the growth of B16.OVA tumors. LC played a critical role in the immunization process because depletion of LC at the time of skin immunization dramatically reduced the tumor-protective effect. The topically applied Ag was presented by skin-derived LC in draining lymph nodes to CD8(+) T cells. Thus, targeting of tumor Ags to LC in vivo is an effective strategy for tumor immunotherapy.     

IL-10 mediates suppression of the CD8 T cell IFN-gamma response to a novel viral epitope in a primed host

Priming to Ag can inhibit subsequent induction of an immune response to a new epitope incorporated into that Ag, a phenomenon referred to as original antigenic sin. In this study, we show that prior immunity to a virus capsid can inhibit subsequent induction of the IFN-gamma effector T cell response to a novel CD8-restricted antigenic epitope associated with the virus capsid. Inhibition does not involve Ab to the virus capsid, as it is observed in animals lacking B cells. CD8-restricted virus-specific T cell responses are not required, as priming to virus without CTL induction is associated with inhibition. However, IL-10(-/-) mice, in contrast to IL-10(+/+) mice, generate CD8 T cell and Ab responses to novel epitopes incorporated into a virus capsid, even when priming to the capsid has resulted in high titer Ab to the capsid. Furthermore, capsid-primed mice, unable to mount a response to a novel epitope in the capsid protein, are nevertheless able to respond to the same novel epitope delivered independently of the capsid. Thus, inhibition of responsiveness to a novel epitope in a virus-primed animal is a consequence of secretion of IL-10 in response to presented Ag, which inhibits local generation of new CD8 IFN-gamma-secreting effector T cells. Induction of virus- or tumor Ag-specific CD8 effector T cells in the partially Ag-primed host may thus be facilitated by local neutralization of IL-10.     

A novel mechanism for complement activation at the surface of B cells following antigen binding

Coligation of CD21 with BCR on the surface of B cells provides a costimulatory signal essential for efficient Ab responses to T-dependent Ags. To achieve this, Ag must be directly linked to C3 fragments, but how this occurs in vivo is not fully understood. Using BCR transgenic mice, we demonstrated that C3 was deposited on the surface of B cells following both high- and moderate-affinity Ag binding. This was dependent on the specific binding of IgM to the BCR-bound Ag and can occur independently of soluble immune complex formation. Based on these data, we propose a novel model in which immune complexes can form directly on the surface of the B cell following Ag binding. This model has implications for our understanding of B lymphocyte activation.     

Anti-idotype and recombinant antigen in immunotherapy of colorectal cancer

The CO17-1A/GA733 antigen (Ag), bound by monoclonal antibodies (MAb) CO17-1A and GA733 that define two different epitopes on the Ag, has proven a useful target in passive and active immunotherapy of colorectal carcinoma (CRC). Previous studies suggest that the antitumor effects demonstrated in MAb-treated patients may be mediated by idiotypic cascades. In approaches to active immunotherapy against the Ag, polyclonal goat and monoclonal rat anti-idiotypic antibodies (Ab2) directed against MAb CO17-1A or GA733 (Ab1) were administered as alum precipitates to 54 patients with CRC (stage Dukes' B, C, and D). The majority of the patients treated with the various Ab2 preparations developed anti-anti-idiotypic antibodies (Ab3) that specifically bound to the CO17-1A or GA733 epitope and shared idiotopes with the corresponding Ab1. Approximately 30% of the patients tested developed specific cellular immunity, i.e., Ag-specific T-cells mediating delayed-type hypersensitivity (DTH) reaction in vivo or proliferating on stimulation with the Ag in vitro. The humoral and cellular immune responses may underlie the clinical responses observed in some of the treated patients. Recently, the CO17-1A/GA733 Ag has been molecularly cloned and expressed in baculo-, adeno-, and vaccinia viruses. In preclinical studies, these recombinant Ag preparations elicited specific humoral immunity (cytotoxic antibodies) and cellular immunity (DTH-reactive and proliferative T-cells), similar to the native Ag. Antibody titers elicited in experimental animals by recombinant Ag were significantly higher than those elicited by Ab2, presumably because Ag expresses numerous epitopes, whereas Ab2 mimics a single epitope. Recombinant CO17-1A/GA733 Ag has potential as a vaccine for CRC patients.     

Aged mice develop protective antitumor immune responses with appropriate costimulation

There is a clear decrease in CD8(+) T cell effector function with aging, a loss once thought to be intrinsic to the CD8(+) T cells. Recent studies suggest, however, that this decline may be a consequence of altered stimulatory signals within the aged lymphoid microenvironment. In this study, we compared the immune responses of young and old mice against the BM-185 pre-B cell lymphoma expressing enhanced GFP (EGFP) as a surrogate tumor Ag. Young animals develop protective immune responses when immunized with BM-185-EGFP, but aged mice do not and ultimately succumb to the tumor. However, expression of CD80 (B7.1) on the BM-185-EGFP (BM-185-EGFP-CD80) results in rejection of the tumor by both young and old animals. Additionally, injection of BM-185-EGFP-CD80 cells in young mice promotes the development of long-lasting memory responses capable of rejecting BM-185 wild-type tumors. Aged animals similarly injected did not develop antitumor memory responses. Interestingly, old animals immunized with the BM-185-EGFP-CD80 cells plus injections of the agonist anti-OX40 mAb did develop long-lasting memory responses capable of rejecting the BM-185 wild-type tumors with the same vigor as the young animals. We show that old mice have the capacity to develop strong antitumor responses and protective memory responses as long as they are provided with efficient costimulation. These results have important implications for the development of vaccination strategies in the elderly, indicating that the aged T cell repertoire can be exploited for the induction of tumor immunity.     

CD8+ T-cell-mediated cross-clade protection in the genital tract following intranasal immunization with inactivated human immunodeficiency virus antigen plus CpG oligodeoxynucleotides

Human immunodeficiency virus (HIV) is a mucosally transmitted infection that rapidly targets and depletes CD4+ T cells in mucosal tissues and establishes a major reservoir for viral persistence in gut-associated lymphoid tissues. Therefore, vaccines designed to prevent HIV infections must induce potent and durable mucosal immune responses, especially in the genital tract. Here we investigated whether intranasal (i.n.) immunization with inactivated gp120-depleted HIV-1 antigen (Ag) plus CpG oligodeoxynucleotide (ODN) as an adjuvant induced local immune responses in the genital tract and cross-clade protection against intravaginal (IVAG) challenge. Lymphocytes isolated from the iliac lymph nodes (ILNs) and genital tracts of female mice i.n. immunized with HIV-1 Ag plus CpG showed significant HIV-specific proliferation and produced significantly higher levels of gamma interferon (IFN-gamma) and beta-chemokines than mice immunized with HIV-1 Ag alone or mixed with non-CpG ODN. CD8+ lymphocytes were dramatically increased in the genital tracts of mice immunized with HIV-1 Ag plus CpG, and protection following IVAG challenge with recombinant vaccinia viruses (rVVs) expressing HIV-1 gag was shown to be CD8 dependent. Finally, cross-clade protection was observed between clades A, C, and G but not B following IVAG challenge with rVVs expressing HIV-1 gag from different clades. These studies provide evidence that mucosal (i.n.) immunization induced strong local T-cell-mediated immune responses in the genital tract and cross-clade protection against IVAG challenge.