Human papillomavirus type 16 virus-like particles expressed in attenuated Salmonella typhimurium elicit mucosal and systemic neutralizing antibodies in mice.

Attenuated strains of Salmonella are attractive live vaccine candidates for eliciting mucosal as well as systemic immune responses. The ability to induce immune responses in the reproductive tract may be critical for the effectiveness of a prophylactic vaccine against genital human papillomaviruses (HPV), which are important etiologic agents in the development of cervical cancer. To examine the potential of a live Salmonella-based vaccine to prevent genital HPV infection, the L1 major capsid protein from HPV type 16 (HPV16) was constitutively expressed in the PhoPc strain of Salmonella typhimurium. As demonstrated by electron microscopy, the L1 protein expressed in these bacteria assembled into virus-like particles (VLPs) that resemble authentic papillomavirus virions. This is the first demonstration that papillomavirus VLPs can self-assemble in prokaryotes. BALB/c mice were immunized with the HPV16 L1 recombinant PhoPc strain by the oral and nasal routes. Despite a low stability of the L1-expressing plasmid in vivo, a double nasal immunization was effective in inducing L1-specific serum antibodies that recognized mainly native, but not disassembled, VLPs. These antibodies effectively neutralized HPV16 pseudotyped virions in an in vitro infectivity assay. Conformationally dependent anti-VLP immunoglobulin A (IgA) and IgG were also detected in oral and vaginal secretions, indicating that potentially protective antibody responses were elicited at mucosal sites. Recombinant attenuated Salmonella expressing HPV capsids may represent a promising vaccine candidate against genital HPV infection.     

Mucosal vaccination with a recombinant Salmonella typhimurium expressing human papillomavirus type 16 (HPV16) L1 virus-like particles (VLPs) or HPV16 VLPs purified from insect cells inhibits the growth of HPV16-expressing tumor cells in mice

Human papillomaviruses, mainly type 16 (HPV16), are responsible for cervical intraepithelial neoplasia, which can lead, in association with other factors, to cervical cancer. Both Salmonella recombinant vaccine strains assembling HPV16 virus-like particles (VLPs) and HPV16 VLPs purified from insect cells are able to induce HPV16 neutralizing antibodies in genital secretions of mice after nasal immunization. Anti-HPV16-specific antibodies in cervical secretions of women may prevent genital infection with HPV16, although this cannot be critically evaluated in the absence of an experimental model for genital papillomavirus infection. Induction of HPV16-specific cell-mediated immunity in the genital mucosa could improve the efficacy of a vaccine and a mucosal route of immunization might be necessary to do so. It has been shown that systemic immunization of mice with purified HPV16 VLPs confers protection against an HPV16-expressing tumor cell challenge through the induction of cytotoxic T-lymphocytes. Using the same C3 tumor model, we show that intranasal immunization of mice with purified HPV16 VLPs in a prophylactic setting also induces anti-tumor immunity. More interestingly, mucosal vaccination of mice with a Salmonella recombinant strain stably expressing HPV16 L1 VLPs also induces anti-tumor immunity in prophylactic as well as in therapeutic settings. Our data suggest that attenuated Salmonella strains expressing chimeric VLPs containing nonstructural viral proteins might be a promising candidate vaccine against cervical cancer by inducing both neutralizing antibodies and cell-mediated immunity.     

Human papillomavirus type 58 L1 virus-like particles purified by two-step chromatography elicit high levels of long-lasting neutralizing antibodies

Human papillomavirus (HPV) type 58 is a high-risk type of HPV frequently detected in cervical cancers, especially in Eastern Asia. There are still no commercially available vaccines against HPV 58 infection. High levels of long-lasting neutralizing antibodies are crucial for long-term protection against HPV infection. Here, we have developed a two-step chromatography strategy and have purified highly pure HPV L1 proteins, which form more homogenous and uniform VLPs than those purified by CsCl ultracentrifugation. Low-dosage immunization with HPV 58 L1 VLPs alone or co-administrated with HPV 16 and HPV 18 L1 VLPs is sufficient to induce high levels of long-lasting neutralizing antibodies in mice. Our results suggest that the highly immunogenic HPV 58 L1 VLPs are a good candidate for use in developing effective vaccines against HPV 58 infection.     

含密码子优化型HPV16 L1基因重组腺病毒的构建及其免疫效果的研究

目的 构建含密码子优化型HPV16L1基因的重组腺病毒,对其经不同接种途径所诱导的系统性及黏膜免疫效果进行研究.方法 使用Admax系统包装重组腺病毒,纯化的腺病毒以不同方式免疫C57BL/6小鼠,间接ELISA及体外中和实验检测免疫小鼠血清及阴道分泌物中的特异性抗体.结果 重组腺病毒滴鼻接种可同时诱导特异性的系统性及黏膜免疫反应,重组腺病毒肌注免疫仅能诱导系统性免疫反应,而阴道黏膜接种不能有效诱导系统性及黏膜免疫反应.结论 成功构建了含密码子优化型HPV 16 L1基因的重组腺病毒,重组腺病毒肌注可诱导高滴度的血清中和抗体,滴鼻接种可同时诱导特异性的系统性及黏膜免疫反应.     

Salmonella enterica serovar Typhi Ty21a expressing human papillomavirus type 16 L1 as a potential live vaccine against cervical cancer and typhoid fever

Human papillomavirus (HPV) vaccines based on L1 virus-like particles (VLPs) can prevent HPV-induced genital neoplasias, the precursors of cervical cancer. However, most cervical cancers occur in developing countries, where the implementation of expensive vaccines requiring multiple injections will be difficult. A live Salmonella-based vaccine could be a lower-cost alternative. We previously demonstrated that high HPV type 16 (HPV16)-neutralizing titers are induced after a single oral immunization of mice with attenuated Salmonella enterica serovar Typhimurium strains expressing a codon-optimized version of HPV16 L1 (L1S). To allow the testing of this type of vaccine in women, we constructed a new L1-expressing plasmid, kanL1S, and tested kanL1S recombinants of three Salmonella enterica serovar Typhi vaccine strains shown to be safe in humans, i.e., Ty21a, the actual licensed typhoid vaccine, and two highly immunogenic typhoid vaccine candidates, Ty800 and CVD908-htrA. In an intranasal mouse model of Salmonella serovar Typhi infection, Ty21a kanL1S was unique in inducing HPV16-neutralizing antibodies in serum and genital secretions, while anti-Salmonella responses were similar to those against the parental Ty21a vaccine. Electron microscopy examination of Ty21a kanL1S lysates showed that L1 assembled in capsomers and capsomer aggregates but not well-ordered VLPs. Comparison to the neutralizing antibody response induced by purified HPV16 L1 VLP immunizations in mice suggests that Ty21a kanL1S may be an effective prophylactic HPV vaccine. Ty21a has been widely used against typhoid fever in humans with a remarkable safety record. These finds encourage clinical testing of Ty21a kanL1S as a combined typhoid fever/cervical cancer vaccine with the potential for worldwide application.     

Induction of cross-neutralizing antibodies by sequential immunization with heterologous papillomavirus L1VLPs and its implications for HPV prophylactic vaccines

Sequential immunization with antigens from different strains of HIV-1, influenza viruses or dengue viruses induced cross-neutralizing antibodies and enhanced the antibody responses against previous antigens. The characteristics of neutralizing antibodies induced by sequential immunization with different types of human papillomavirus (HPV) L1 virus-like particles (L1VLPs) are unclear. In this study, mice were primed with one or two types (HPV-16 or HPV16/18) of L1VLPs, then boosted sequentially with HPV6/18/45/11/31/58 or HPV6/45/11/31/58 L1VLPs, and sera were analyzed with HPV pseudovirus-based neutralization assay. The results showed that neutralizing activities against earlier immunized vaccine types were enhanced gradually by subsequent immunizations, and low levels of neutralizing activities against nonvaccine types (HPV33/35/52/59/68) were also observed. After absorbing the immune sera with vaccine-type (HPV16/18/45) L1VLPs, neutralizing activities against tested priming and boosting types (HPV16/18/58) decreased significantly, and that against nonvaccine type (HPV-33) was also partially eliminated. Moreover, neutralizing activities against vaccine types (HPV16/58) were significantly reduced after absorbing with nonvaccine-type VLPs (HPV33/52). These data suggest that cross-neutralizing epitopes exist among different HPV L1VLPs. The cross-neutralizing activities against nonvaccine types and the enhanced neutralizing activities against earlier immunized vaccine types may result from sequential boosting with these cross-neutralizing epitopes. These observations support early vaccination with more types of L1VLPs derived from HPVs that cause a serious threat to the population.     

Induction of Multiepitopic and Long-Lasting Immune Responses Against Tumour Antigens by Immunization with Peptides, DNA and Recombinant Adenoviruses Expressing Minigenes

The development of immunization strategies to induce strong and multiepitopic T-cell responses against tumour antigens is needed for anti-tumour immunotherapy. However, a common finding after immunization with complex antigens is the preferential induction of immune responses against immunodominant epitopes. In this study, with the aim of inducing multiepitopic responses against several common tumour antigens, we have designed a minigene construct encoding four human leucocyte antigen (HLA)-A2-restricted epitopes belonging to tumour antigens CEA (CEA-691 and CEA-571), MAGE2 (MAGE2-157) and MAGE3 (MAGE3-112), as well as the universal PADRE epitope recognized by T helper lymphocytes. To optimize the activation of immune responses against these epitopes, we have used different antigen formats (short peptides encompassing individual epitopes and DNA plasmids or adenoviral constructs expressing the minigene) in single or combined immunization schedules. A single immunization with either DNA plasmid or recombinant adenovirus induced a monospecific immune response against the immunodominant epitope CEA-571, whereas immunization with the peptide pool induced responses against all epitopes. Combination of peptide priming followed by a boost with the plasmid and the recombinant adenovirus expressing the minigene induced stronger, multi-specific and long-lasting immune responses, overcoming the immunodominance imposed by the main T-cell epitope. Moreover, these combined immunization strategies were able to induce responses that were able to recognize Mel624 HLA-A2⁺ tumour cells expressing MAGE2. These results suggest that heterologous immunization strategies combining peptides and DNA or recombinant adenoviruses can be useful to broaden the specificity and enhance the efficacy of subunit vaccines.     

Immunization with an HPV-16 L1-based chimeric virus-like particle containing HPV-16 E6 and E7 epitopes elicits long-lasting prophylactic and therapeutic efficacy in an HPV-16 tumor mice model

HPV L1-based virus-like particles vaccines (VLPs) efficiently induce temporary prophylactic activity through the induction of neutralizing antibodies; however, VLPs that can provide prophylactic as well as therapeutic properties for longer periods of time are needed. For this purpose, we generated a novel HPV 16 L1-based chimeric virus-like particle (cVLP) produced in plants that contains a string of T-cell epitopes from HPV 16 E6 and E7 fused to its C-terminus. In the present study, we analyzed the persistence of specific IgG antibodies with neutralizing activity induced by immunization with these cVLPs, as well as their therapeutic potential in a tumor model of C57BL/6 mice. We observed that these cVLPs induced persistent IgG antibodies for over 12 months, with reactivity and neutralizing activity for VLPs composed of only the HPV-16 L1 protein. Efficient protection for long periods of time and inhibition of tumor growth induced by TC-1 tumor cells expressing HPV-16 E6/E7 oncoproteins, as well as significant tumor reduction (57 %), were observed in mice immunized with these cVLPs. Finally, we discuss the possibility that chimeric particles of the type described in this work may be the basis for developing HPV prophylactic and therapeutic vaccines with high efficacy.     

Expression of hepatitis B virus antigens in attenuated Salmonellae for oral immunization

The aim of our work is to identify hepatitis B virus antigens that can be stably expressed in attenuated Salmonellae and elicit protective immune responses as live oral route vaccines. As a first carrier system, we expressed T-cell and B-cell epitopes of hepatitis B virus as fusion proteins with the non-toxic subunit B (LT-B) in attenuated Salmonellae. These recombinant Salmonellae elicited anti-LT-B T- and B-cell immune responses and anti-HBV nucleocapsid antigen (HBcAg) T-cell responses when fed to mice. To combine the protective potential and the high immunogenicity of HBc with the induction of virus neutralizing antibodies to HBV surface antigen, we constructed vectors expressing hybrid HBc/pre-S particles in which the pre-S epitopes were surface-exposed. With one of these vectors, stable constitutive high level expression of hybrid HBc/pre-S2 particles was achieved in several attenuated Salmonella strains. When recombinant Salmonellae expressing such hybrid HBc/pre-S2 fusion proteins were fed to mice, the animals developed high titres of anti-HBcAg-specific serum IgG after a single or multiple oral immunizations, depending on the strain used as a carrier. In addition, lower titered antibodies against the pre-S2 antibody-binding sites were elicited. This is the first HBV antigen eliciting high-titered immune responses after a single oral immunization in recombinant Salmonellae. The immunogenicity of periplasmic LT-B and cytoplasmic HBc/pre-S2 shows that surface exposure of a foreign antigen is not a prerequisite for its immunogenicity in live attenuated Salmonellae.     

Preventative and therapeutic vaccines for cervical cancer

"High-risk" genotypes of the human papillomavirus (HPV), most commonly HPV genotype 16, are the primary etiologic agents of cervical cancer. Indeed HPV DNA is detected in 99% of cervical carcinomas. Thus, cervical cancer and other HPV-associated malignancies might be prevented or treated by the induction of the appropriate viral-antigen-specific immune responses. Transmission of papillomavirus may be prevented by the generation of antibodies to capsid proteins L1 and L2 that neutralize viral infection. HPV L1 virus-like particles (VLPs) show great promise as prophylactic HPV vaccines in ongoing clinical trials but L2-based preventative vaccines have yet to be tested in patients. Since the capsid proteins are not expressed at detectable levels by infected basal keratinocytes or in HPV-transformed cells, therapeutic vaccines generally target the nonstructural early viral antigens. Two HPV oncogenic proteins, E6 and E7, are critical to the induction and maintenance of cellular transformation and are co-expressed in the majority of HPV-containing carcinomas. Although other early viral antigens show promise for vaccination against papillomas, therapeutic vaccines targeting E6 and E7 may provide the best opportunity to control HPV-associated malignancies. Various candidate therapeutic HPV vaccines are currently being tested whereby E6 and/or E7 are administered in live vectors, as peptides or proteins, in nucleic acid form, as components of chimeric VLPs, or in cell-based vaccines. Encouraging results from experimental vaccination systems in animal models have led to several prophylactic and therapeutic vaccine clinical trials. Should this new generation of HPV preventative and therapeutic vaccines function in patients as demonstrated in animal models, oncogenic HPV infection and its associated malignancies could be controlled by vaccination. Importantly, recent advances in HPV detection and continued improvements in screening further enhance our opportunities to systematically eradicate HPV-associated malignancy.     

Progress in prophylactic and therapeutic vaccines for human papillomavirus infection

Virus-like particle (VLP) subunit vaccines composed of the major capsid protein L1 of the genital human papillomaviruses (HPVs) are now in Phase III clinical trials. The vaccines are immunogenic and safe and early results indicate efficacy. VLPs induce strong cell-mediated as well as humoral immune responses and chimeric VLPs including an HPV early protein may have therapeutic potential. Polynucleotide and recombinant viral vaccines encoding nonstructural viral proteins show therapeutic and prophylactic efficacy in animal models and are candidate immunotherapies for established low-grade benign genital infections. Vaccines designed to elicit cytotoxic T-lymphocytes specific for the HPV oncoproteins E6 and E7 show immunogenicity and efficacy in transplantable tumor models in rodents. In Phase I and II trials these vaccines are immunogenic and safe but show limited efficacy.     

A deletion and point mutation study of the human papillomavirus type 16 major capsid gene

Recombinant human papillomavirus (HPV) virus-like particles (VLPs) made from the major capsid protein L1 are promising vaccine candidates for use as vaccines against genital and other HPV infections, and particularly against HPV-16. However, HPV-16 genotype variants have different binding affinities for neutralising mouse Mabs raised against HPV-16 L1 VLPs. This paper analyses, using a panel of well-characterised Mabs, the effects on the antigenicity of various C- and N-terminal deletants of HPV-16 L1 made in insect cells via recombinant baculovirus, of an A-->T mutation at residue 266 (A266T), and of a C-->G mutation at conserved position 428 (C428G). The effects of these changes on assembly of the variant L1s were studied by electron microscopy. Binding of Mab H16:E70 to A266T was reduced by almost half in comparison to wild type L1. Retention of the C-terminal region 428-483 was critical for the binding of conformation-specific Mabs (H16:V5, H16:E70, H16:U4 and H16:9A) whereas deletion of the nuclear localisation signal (NLS) or the C428G mutation or an N-terminal deletion (residues 2-9) did not affect the antigenicity. The N-terminal deletion resulted in a mixed population of 30 and 55nm VLPs, which differs from the same construct expressed in Escherichia coli, whereas pentamer aggregates resulted from deletion of the 428-465 region or the C428G mutation. The results have implications both for considering use of single-genotype HPV vaccines, and for design of novel second-generation vaccines.     

Mycobacterium bovis BCG priming induces a strong potentiation of the antibody response induced by recombinant BCG expressing a foreign antigen.

Several recent studies have demonstrated that strong cellular or humoral immune responses can be induced against foreign antigens expressed by recombinant Mycobacterium bovis BCG. It has therefore been suggested that BCG could represent one of the best candidate vectors for live recombinant vaccines. However, a large percentage of the human population has been immunized by BCG, and this priming could modify the immune response to future recombinant BCG vaccines. In the present study, we have therefore compared the immune responses induced in naive and BCG-primed mice by two recombinant BCG vaccines expressing either beta-galactosidase or human immunodeficiency virus type 1 Nef antigens. Our results demonstrated that BCG priming limits the growth of recombinant BCG in mouse spleen or lymph nodes. This reduction in BCG growth was associated with decreased proliferative responses against Nef or beta-galactosidase antigens. This suppression, however, never exceeded 50%. Interestingly, in contrast to these reduced T-cell responses, BCG-primed mice developed high levels of anti-beta-galactosidase antibodies after immunization with recombinant BCG expressing this antigen. This stimulation of antibody responses was not due to polyclonal stimulation or to a nonspecific adjuvant effect of BCG. The isotypic patterns of anti-beta-galactosidase antibody responses induced by the recombinant BCG were similar in naive and BCG-primed mice. These results indicate that priming with BCG will not be a limitation for the use of recombinant BCG vaccines in humans.     

Comparison of systemic and mucosal vaccination: impact on intravenous and rectal SIV challenge

Mucosal tissues are the primary route of transmission for most respiratory and sexually transmitted diseases, including human immunodeficiency virus. We aimed to generate strong mucosal immune responses to simian immunodeficiency virus (SIV) in rhesus macaques by targeting recombinant adenovirus serotype 5 (rAd5) to the lung. The immunogenicity and efficacy of aerosol (AE) vaccination was compared with intramuscular (IM) delivery in either an intravenous (IV) or intrarectal (IR) SIV(mac251) challenge model. Aerosolized rAd5 induced strong cellular responses in the lung and systemic humoral responses equivalent to IM. Strikingly, all immunization groups controlled acute viremia in the IV challenge model by 1-2 logs. By contrast, after IR challenge, only peak viremia was reduced by immunization, with no significant effect on SIV infection acquisition rate or mucosal CD4(+) T-cell preservation. Improved disease outcome was associated with pre-challenge cellular and humoral responses, while post-challenge T-cell responses were highly correlated with viremia control. The similar outcomes achieved by systemic and airway mucosal immunization support AE delivery as a safe, effective, and less invasive alternative to parenteral vaccination.     

Chimeric human papilloma virus-simian/human immunodeficiency virus virus-like-particle vaccines: immunogenicity and protective efficacy in macaques

Vaccines to efficiently block or limit sexual transmission of both HIV and human papilloma virus (HPV) are urgently needed. Chimeric virus-like-particle (VLP) vaccines consisting of both multimerized HPV L1 proteins and fragments of SIV gag p27, HIV-1 tat, and HIV-1 rev proteins (HPV-SHIV VLPs) were constructed and administered to macaques both systemically and mucosally. An additional group of macaques first received a priming vaccination with DNA vaccines expressing the same SIV and HIV-1 antigens prior to chimeric HPV-SHIV VLP boosting vaccinations. Although HPV L1 antibodies were induced in all immunized macaques, weak antibody or T cell responses to the chimeric SHIV antigens were detected only in animals receiving the DNA prime/HPV-SHIV VLP boost vaccine regimen. Significant but partial protection from a virulent mucosal SHIV challenge was also detected only in the prime/boosted macaques and not in animals receiving the HPV-SHIV VLP vaccines alone, with three of five prime/boosted animals retaining some CD4+ T cells following challenge. Thus, although some immunogenicity and partial protection was observed in non-human primates receiving both DNA and chimeric HPV-SHIV VLP vaccines, significant improvements in vaccine design are required before we can confidently proceed with this approach to clinical trials.     

The Nature of the Attenuation of Salmonella typhimurium Strains Expressing Human Papillomavirus Type 16 Virus-Like Particles Determines the Systemic and Mucosal Antibody Responses in Nasally Immunized Mice

We have recently shown by using a recombinant Salmonella typhimurium PhoPc strain in mice the feasibility of using a Salmonella-based vaccine to prevent infection by the genital human papillomavirus type 16 (HPV16). Here, we compare the HPV16-specific antibody responses elicited by nasal immunization with recombinant S. typhimurium strains harboring attenuations that, in contrast to PhoPc, are suitable for human use. For this purpose, chi4989 (Deltacya Deltacrp) and chi4990 [Deltacya Delta(crp-cdt)] were constructed in the ATCC 14028 genetic background, and comparison was made with the isogenic PhoPc and PhoP- strains. Although the levels of expression of HPV16 virus-like particle (VLP) were similar in all strains, only PhoPc HPV16 induced sustained specific antibody responses after nasal immunization, while all strains induced high antibody responses with a single nasal immunization when an unrelated viral hepatitis B core antigen was expressed. The level of the specific antibody responses induced did not correlate with the number of recombinant bacteria surviving in various organs 2 weeks after immunization. Our data suggest that the immunogenicity of attenuated Salmonella vaccine strains does not correlate with either the number of persisting bacteria after immunization or the levels of in vitro expression of the antigen carried. Rather, the PhoPc phenotype appears to provide the unique ability in Salmonella to induce immune responses against HPV16 VLPs.     

A single immunization with a recombinant canine adenovirus expressing the rabies virus G protein confers protective immunity against rabies in mice

Rabies vaccines based on live attenuated rabies viruses or recombinant pox viruses expressing the rabies virus (RV) glycoprotein (G) hold the greatest promise of safety and efficacy, particularly for oral immunization of wildlife. However, while these vaccines induce protective immunity in foxes, they are less effective in other animals, and safety concerns have been raised for some of these vaccines. Because canine adenovirus 2 (CAV2) is licensed for use as a live vaccine for dogs and has an excellent efficacy and safety record, we used this virus as an expression vector for the RVG. The recombinant CAV2-RV G produces virus titers similar to those produced by wild-type CAV2, indicating that the RVG gene does not affect virus replication. Comparison of RVG expressed by CAV2-RV G with that of vaccinia-RV G recombinant virus (V-RG) revealed similar amounts of RV G on the cell surface. A single intramuscular or intranasal immunization of mice with CAV2-RVG induced protective immunity in a dose-dependent manner, with no clinical signs or discomfort from the virus infection regardless of the route of administration or the amount of virus.     

Definition of linear antigenic regions of the HPV16 L1 capsid protein using synthetic virion-like particles.

Mice of three haplotypes (H-2d, H-2b, and H-2d/b) were immunized with synthetic HPV16 virus-like particles (VLPs), produced using a vaccinia virus doubly recombinant for the L1 and L2 proteins of HPV16. The resultant anti-VLP antisera recognized HPV16 capsids by ELISA assay and baculovirus recombinant HPV16 L1 and L2 protein on immunoblot. Overlapping peptides corresponding to the HPV16 L1 amino acid sequence were used to define the immunoreactive regions of the L1 protein. The majority of the L1 peptides were reactive with IgG from the mice immunized with the synthetic HPV16 capsids. A computer algorithm predicted seven B epitopes in HPV16 L1, five of which lay within peptides strongly reactive with the murine antisera. The murine anti-VLP antisera failed to react with the two peptides recognized by anti-HPV16L1 monoclonal antibodies raised by others against recombinant L1 fusion protein. We conclude that the immunoreactive epitopes of HPV16 defined using virus-like particles differ significantly from those defined using recombinant HPV16 L1 fusion proteins, which implies that such fusion proteins may not be the antigens to look for HPV16L1 specific immune responses in HPV-infected patients.     

DNA prime Listeria boost induces a cellular immune response to SIV antigens in the rhesus macaque model that is capable of limited suppression of SIV239 viral replication

DNA vaccines and recombinant Listeria monocytogenes that express and secrete SIV Gag and Env antigens were combined in a nonhuman primate prime-boost immunogenicity study followed by a challenge with SIV239. We report that recombinant DNA vaccine delivered intramuscularly, and recombinant L. monocytogenes delivered orally each individually have the ability to induce CD8+ and CD4+ T cell immune responses in a nonhuman primate. Four rhesus monkeys were immunized at weeks 0, 4, 8, and 12 with the pCSIVgag and pCSIVenv DNA plasmids and boosted with SIV expressing L. monocytogenes vaccines at weeks 16, 20, and 28. Four rhesus monkeys received only the L. monocytogenes vaccines at weeks 16, 20, and 28. A final group of monkeys served as a control group. Blood samples were taken before vaccination and 2 weeks post each injection and analyzed by ELISPOT for CD4+ and CD8+ T cell responses. Moderate vaccine induced SIV-specific cellular immune responses were observed following immunization with either DNA or L. monocytogenes vectors. However, the SIV antigen-specific immune responses were significantly increased when Rhesus macaques were primed with SIV DNA vaccines and boosted with the SIV expressing L. monocytogenes vectors. In addition, the combined vaccine was able to impact SIV239 viral replication following an intrarectal challenge. This study demonstrates for the first time that oral L. monocytogenes can induce a cellular immune response in a nonhuman primate and is able to enhance the efficacy of a DNA vaccine as well as provide modest protection against SIV239 challenge.