The Use of Directed Evolution to Create a Stable and Immunogenic Recombinant BCG Expressing a Modified HIV-1 Gag Antigen

Numerous features make Mycobacterium bovis BCG an attractive vaccine vector for HIV. It has a good safety profile, it elicits long-lasting cellular immune responses and in addition manufacturing costs are affordable. Despite these advantages it is often difficult to express viral antigens in BCG, which results in genetic instability and low immunogenicity. The aim of this study was to generate stable recombinant BCG (rBCG) that express high levels of HIV antigens, by modification of the HIV genes. A directed evolution process was applied to recombinant mycobacteria that expressed HIV-1 Gag fused to the green fluorescent protein (GFP). Higher growth rates and increased GFP expression were selected for. Through this process a modified Gag antigen was selected. Recombinant BCG that expressed the modified Gag (BCG[pWB106] and BCG[pWB206]) were more stable, produced higher levels of antigen and grew faster than those that expressed the unmodified Gag (BCG[pWB105]). The recombinant BCG that expressed the modified HIV-1 Gag induced 2 to 3 fold higher levels of Gag-specific CD4 T cells than those expressing the unmodified Gag (BCG[pWB105]). Mice primed with 10⁷ CFU BCG[pWB206] and then boosted with MVA-Gag developed Gag-specific CD8 T cells with a frequency of 1343±17 SFU/10⁶ splenocytes, 16 fold greater than the response induced with MVA-Gag alone. Levels of Gag-specific CD4 T cells were approximately 5 fold higher in mice primed with BCG[pWB206] and boosted with MVA-Gag than in those receiving the MVA-Gag boost alone. In addition mice vaccinated with BCG[pWB206] were protected from a surrogate vaccinia virus challenge.     

A pantothenate auxotroph of Mycobacterium tuberculosis is highly attenuated and protects mice against tuberculosis

With the advent of HIV and the widespread emergence of drug-resistant strains of Mycobacterium tuberculosis, newer control strategies in the form of a better vaccine could decrease the global incidence of tuberculosis. A desirable trait in an effective live attenuated vaccine strain is an ability to persist within the host in a limited fashion in order to produce important protective antigens in vivo. Attenuated M. tuberculosis vaccine candidates have been constructed by deleting genes required for growth in mice. These candidate vaccines did not elicit adequate protective immunity in animal models, due to their inability to persist sufficiently long within the host tissues. Here we report that an auxotrophic mutant of M. tuberculosis defective in the de novo biosynthesis of pantothenic acid (vitamin B5) is highly attenuated in immunocompromised SCID mice and in immunocompetent BALB/c mice. SCID mice infected with the pantothenate auxotroph survived significantly longer (250 days) than mice infected with either bacille Calmette-Guerin (BCG) vaccine or virulent M. tuberculosis (77 and 35 days, respectively). Subcutaneous immunization with this auxotroph conferred protection in C57BL/6J mice against an aerosol challenge with virulent M. tuberculosis, which was comparable with that afforded by BCG vaccination. Our findings highlight the importance of de novo pantothenate biosynthesis in limiting the intracellular survival and pathogenesis of M. tuberculosis without reducing its immunogenicity in vaccinated mice.     

Newcastle disease virus expressing a dendritic cell-targeted HIV gag protein induces a potent gag-specific immune response in mice

Viral vaccine vectors have emerged as an attractive strategy for the development of a human immunodeficiency virus (HIV) vaccine. Recombinant Newcastle disease virus (rNDV) stands out as a vaccine vector since it has a proven safety profile in humans, it is a potent inducer of both alpha interferon (IFN-α) and IFN-β) production, and it is a potent inducer of dendritic cell (DC) maturation. Our group has previously generated an rNDV vector expressing a codon-optimized HIV Gag protein and demonstrated its ability to induce a Gag-specific CD8(+) T cell response in mice. In this report we demonstrate that the Gag-specific immune response can be further enhanced by the targeting of the rNDV-encoded HIV Gag antigen to DCs. Targeting of the HIV Gag antigen was achieved by the addition of a single-chain Fv (scFv) antibody specific for the DC-restricted antigen uptake receptor DEC205 such that the DEC205 scFv-Gag molecule was encoded for expression as a fusion protein. The vaccination of mice with rNDV coding for the DC-targeted Gag antigen induced an enhanced Gag-specific CD8(+) T cell response and enhanced numbers of CD4(+) T cells and CD8(+) T cells in the spleen relative to vaccination with rNDV coding for a nontargeted Gag antigen. Importantly, mice vaccinated with the DEC205-targeted vaccine were better protected from challenge with a recombinant vaccinia virus expressing the HIV Gag protein. Here we demonstrate that the targeting of the HIV Gag antigen to DCs via the DEC205 receptor enhances the ability of an rNDV vector to induce a potent antigen-specific immune response.     

Vaginal protection and immunity after oral immunization of mice with a novel vaccine strain of Listeria monocytogenes expressing human immunodeficiency virus type 1 gag

Natural transmission of human immunodeficiency virus (HIV) occurs at mucosal surfaces. During acute infection, intestinal and other mucosae are preferential sites of virus replication and rapidly become depleted of CD4(+) T cells. Therefore, mucosal immunity may be critical to control both initial infection and the massive early spread of virus. An attenuated D-alanine-requiring strain of the oral intracellular microorganism Listeria monocytogenes expressing HIV type 1 gag was shown to induce protective cell-mediated immunity in mice against viruses that express HIV gag when immunization occurs in the presence of a transient supply of D-alanine. In this study, we examined the efficacy of new attenuated strains that are able to synthesize d-alanine from a heterologous dal gene tightly regulated by an actA-promoted resolvase recombination system. In the absence of d-alanine, Gag-specific cytotoxic T lymphocytes (CTLs) were induced systemically after intravenous immunization, and one strain, Lmdd-gag/pARS, induced strong dose-dependent Gag-specific CTLs after oral immunization. A significant level of Gag-specific CD8(+) T cells was induced in the mucosal-associated lymphoid tissues (MALTs). Upon intravaginal challenge of these orally immunized mice with recombinant vaccinia virus (rVV) expressing HIV gag, gamma interferon- and tumor necrosis factor alpha-secreting Gag-specific CD8(+) T cells were dramatically increased in the spleen and MALTs. Oral immunization with Lmdd-gag/pARS led to complete protection against vaginal challenge by a homologous clade B gag-expressing rVV. In addition, strong cross-clade protection was seen against clades A and C and partial protection against clade G gag-expressing rVV. These results suggest that Lmdd-gag/pARS may be considered as a novel vaccine candidate for use against HIV/AIDS.     

Human immunodeficiency virus type 1 gag-specific mucosal immunity after oral immunization with papillomavirus pseudoviruses encoding gag

Mucosal surfaces are the primary portals for human immunodeficiency virus (HIV) transmission. Because systemic immunization, in general, does not induce effective mucosal immune responses, a mucosal HIV vaccine is urgently needed. For this study, we developed papillomavirus pseudoviruses that express HIV-1 Gag. The pseudoviruses are synthetic, nonreplicating viruses, yet they can produce antigens for a long time in the immune system. Here we show that oral immunization of mice by the use of papillomavirus pseudoviruses encoding Gag generated mucosal and systemic Gag-specific cytotoxic T lymphocytes that effectively lysed Gag-expressing target cells. Furthermore, the pseudoviruses generated Gag-specific gamma interferon-producing T cells and serum immunoglobulin G (IgG) and mucosal IgA. In contrast, oral immunization with plasmid DNA encoding HIV-1 Gag did not induce specific immune responses. Importantly, oral immunization with the pseudoviruses induced Gag-specific memory cytotoxic T lymphocytes and protected mice against a rectal mucosal challenge with a recombinant vaccinia virus expressing HIV-1 Gag. Thus, papillomavirus pseudoviruses encoding Gag are a promising mucosal vaccine against AIDS.     

HIV gag基因修饰在不同载体系统中对免疫效果的影响

目的:了解gag基因修饰前后在不同载体系统中的表达水平差异对免疫效果的影响,为确定HIV疫苗中能诱发较高水平细胞免疫的Gag靶抗原奠定实验基础。方法:将含有优化前后gag基因的HIV-1 DNA(pVRC)疫苗和重组痘苗病毒(rVV)载体疫苗单独或联合免疫BALB/c小鼠,利用IFN-γ酶联免疫斑点法和胞内细胞因子染色检测各组的细胞免疫效果,ELISA检测体液免疫水平,分别比较基因优化前后及在不同载体内的Gag诱发的免疫效果。结果:DNA疫苗中gag基因修饰后细胞免疫反应由472提高至925 SFC/106MNC,抗体滴度随免疫次数增加而提高,基因修饰后第二针的抗体水平由104.2提高至105.3,三针后则没有差别;而以rVV为载体的疫苗基因修饰前后细胞免疫反应(~320 SFC/106MNC)和抗体水平(~104.4)均没有差异。2种疫苗联合免疫均可显著提高Gag修饰前后在小鼠体内的免疫效果,基因修饰后细胞免疫反应由1700提高至2100 SFC/106MNC,抗体水平则没有差别。结论:gag基因修饰明显提高常规DNA疫苗免疫效果,并可进一步提高联合免疫效果,但对rVV疫苗单独免疫效果无明显影响。     

Mycobacterial codon optimization enhances antigen expression and virus-specific immune responses in recombinant Mycobacterium bovis bacille Calmette-Guérin expressing human immunodeficiency virus type 1 Gag

Although its potential for vaccine development is already known, the introduction of recombinant human immunodeficiency virus (HIV) genes to Mycobacterium bovis bacille Calmette-Guérin (BCG) has thus far elicited only limited responses. In order to improve the expression levels, we optimized the codon usage of the HIV type 1 (HIV-1) p24 antigen gene of gag (p24 gag) and established a codon-optimized recombinant BCG (rBCG)-p24 Gag which expressed a 40-fold-higher level of p24 Gag than did that of nonoptimized rBCG-p24 Gag. Inoculation of mice with the codon-optimized rBCG-p24 Gag elicited effective immunity, as evidenced by virus-specific lymphocyte proliferation, gamma interferon ELISPOT cell induction, and antibody production. In contrast, inoculation of animals with the nonoptimized rBCG-p24 Gag induced only low levels of immune responses. Furthermore, a dose as small as 0.01 mg of the codon-optimized rBCG per animal proved capable of eliciting immune responses, suggesting that even low doses of a codon-optimized rBCG-based vaccine could effectively elicit HIV-1-specific immune responses.     

Immunogenicity of DNA and Recombinant Sendai Virus Vaccines Expressing the HIV-1 gag Gene

Combinations of DNA and recombinant-viral-vector based vaccines are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. It was found that Gag-specific cytotoxic lymphocyte (CTL) responses were associated with lowering viremia in an untreated HIV-1 infected cohort. The main objectives of our studies were the construction of DNA and recombinant Sendal virus vector (rSeV) vaccines containing a gag gene from the prevalent Thailand subtype B strain in China and trying to use these vaccines for therapeutic and prophylactic vaccines. The candidate plasmid DNA vaccine pcDNA3.1( )-gag and recombinant Sendai virus vaccine (rSeV-gag) were constructed separately. It was verified by Western blotting analysis that both DNA and rSeV-gag vaccines expressed the HIV-1 Gag protein correctly and efficiently. Balb/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gag-specific CTL responses and antibody levels were detected by intracellular cytokine staining assay and enzyme-linked immunosorbant assay (ELISA) respectively. Combined vaccines in a DNA prime/rSeV-gag boost vaccination regimen induced the strongest and most long-lasting Gag-specific CTL and antibody responses. It maintained relatively high levels even 9 weeks post immunization. This data indicated that the prime-boost regimen with DNA and rSeV-gag vaccines may offer promising HIV vaccine regimens.     

Lysine auxotrophy combined with deletion of the SecA2 gene results in a safe and highly immunogenic candidate live attenuated vaccine for tuberculosis

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a major global health problem, despite the widespread use of the M. bovis Bacille Calmette-Guerin (BCG) vaccine and the availability of drug therapies. In recent years, the high incidence of coinfection of M. tuberculosis and HIV, as well as escalating problems associated with drug resistance, has raised ominous concerns with regard to TB control. Vaccination with BCG has not proven highly effective in controlling TB, and also has been associated with increasing concerns about the potential for the vaccine to cause disseminated mycobacterial infection in HIV infected hosts. Thus, the development of an efficacious and safe TB vaccine is generally viewed as a critical to achieving control of the ongoing global TB pandemic. In the current study, we have analyzed the vaccine efficacy of an attenuated M. tuberculosis strain that combines a mutation that enhances T cell priming (ΔsecA2) with a strongly attenuating lysine auxotrophy mutation (ΔlysA). The ΔsecA2 mutant was previously shown to be defective in the inhibition of apoptosis and markedly increased priming of antigen-specific CD8(+) T cells in vivo. Similarly, the ΔsecA2ΔlysA strain retained enhanced apoptosis and augmented CD8(+) T cell stimulatory effects, but with a noticeably improved safety profile in immunosuppressed mice. Thus, the M. tuberculosis ΔsecA2ΔlysA mutant represents a live attenuated TB vaccine strain with the potential to deliver increased protection and safety compared to standard BCG vaccination.     

Priming-boosting vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin and a nonreplicating vaccinia virus recombinant leads to long-lasting and effective immunity

Virus-specific T-cell responses can limit immunodeficiency virus type 1 (HIV-1) transmission and prevent disease progression and so could serve as the basis for an affordable, safe, and effective vaccine in humans. To assess their potential for a vaccine, we used Mycobacterium bovis bacillus Calmette-Guérin (BCG)-Tokyo and a replication-deficient vaccinia virus strain (DIs) as vectors to express full-length gag from simian immunodeficiency viruses (SIVs) (rBCG-SIVgag and rDIsSIVgag). Cynomolgus macaques were vaccinated with either rBCG-SIVgag dermally as a single modality or in combination with rDIsSIVgag intravenously. When cynomologus macaques were primed with rBCG-SIVgag and then boosted with rDIsSIVgag, high levels of gamma interferon (IFN-gamma) spot-forming cells specific for SIV Gag were induced. This combination regimen elicited effective protective immunity against mucosal challenge with pathogenic simian-human immunodeficiency virus for the 1 year the macaques were under observation. Antigen-specific intracellular IFN-gamma activity was similarly induced in each of the macaques with the priming-boosting regimen. Other groups receiving the opposite combination or the single-modality vaccines were not effectively protected. These results suggest that a recombinant M. bovis BCG-based vector may have potential as an HIV/AIDS vaccine when administered in combination with a replication-deficient vaccinia virus DIs vector in a priming-boosting strategy.     

Mucosal priming of simian immunodeficiency virus-specific cytotoxic T-lymphocyte responses in rhesus macaques by the Salmonella type III secretion antigen delivery system

Nearly all human immunodeficiency virus (HIV) infections are acquired mucosally, and the gut-associated lymphoid tissues are important sites for early virus replication. Thus, vaccine strategies designed to prime virus-specific cytotoxic T lymphocyte (CTL) responses that home to mucosal compartments may be particularly effective at preventing or containing HIV infection. The Salmonella type III secretion system has been shown to be an effective approach for stimulating mucosal CTL responses in mice. We therefore tested DeltaphoP-phoQ attenuated strains of Salmonella enterica serovar Typhimurium and S. enterica serovar Typhi expressing fragments of the simian immunodeficiency virus (SIV) Gag protein fused to the type III-secreted SopE protein for the ability to prime virus-specific CTL responses in rhesus macaques. Mamu-A*01(+) macaques were inoculated with three oral doses of recombinant Salmonella, followed by a peripheral boost with modified vaccinia virus Ankara expressing SIV Gag (MVA Gag). Transient low-level CTL responses to the Mamu-A*01 Gag(181-189) epitope were detected following each dose of SALMONELLA: After boosting with MVA Gag, strong Gag-specific CTL responses were consistently detected, and tetramer staining revealed the expansion of Gag(181-189)-specific CD8(+) T-cell responses in peripheral blood. A significant percentage of the Gag(181-189)-specific T-cell population in each animal also expressed the intestinal homing receptor alpha4beta7. Additionally, Gag(181-189)-specific CD8(+) T cells were detected in lymphocytes isolated from the colon. Yet, despite these responses, Salmonella-primed/MVA-boosted animals did not exhibit improved control of virus replication following a rectal challenge with SIVmac239. Nevertheless, this study demonstrates the potential of mucosal priming by the Salmonella type III secretion system to direct SIV-specific cellular immune responses to the gastrointestinal mucosa in a primate model.     

Induction of human immunodeficiency virus (HIV)-specific CD8 T-cell responses by Listeria monocytogenes and a hyperattenuated Listeria strain engineered to express HIV antigens

Induction of cell-mediated immunity may be essential for an effective AIDS vaccine. Listeria monocytogenes is an attractive bacterial vector to elicit T-cell immunity to human immunodeficiency virus (HIV) because it specifically infects monocytes, key antigen-presenting cells, and because natural infection originates at the mucosa. Immunization with recombinant L. monocytogenes has been shown to protect mice from lymphocytic choriomeningitis virus, influenza virus, and tumor inoculation. L. monocytogenes expressing HIV gag elicits sustained high levels of Gag-specific cytotoxic T lymphocytes (CTLs) in mice. We have examined the ability of Listeria to infect human monocytes and present HIV antigens to CD8 T lymphocytes of HIV-infected donors to induce a secondary T-cell immune response. Using this in vitro vaccination protocol, we show that L. monocytogenes expressing the HIV-1 gag gene efficiently provides a strong stimulus for Gag-specific CTLs in HIV-infected donor peripheral blood mononuclear cells. Listeria expressing Nef also elicits a secondary in vitro anti-Nef CTL response. Since L. monocytogenes is a pathogen, before it can be seriously considered as a human vaccine vector, safety concerns must be addressed. We therefore have produced a highly attenuated strain of L. monocytogenes that requires D-alanine for viability. The recombinant bacteria are attenuated at least 10(5)-fold. We show that when these hyperattenuated bacteria are engineered to express HIV-1 Gag, they are at least as efficient at stimulating Gag-specific human CTLs in vitro as wild-type recombinants. These results suggest that attenuated Listeria is an attractive candidate vaccine vector to induce T-cell immunity to HIV in humans.     

Comparison of Heterologous Prime-Boost Strategies against Human Immunodeficiency Virus Type 1 Gag Using Negative Stranded RNA Viruses

This study analyzed a heterologous prime-boost vaccine approach against HIV-1 using three different antigenically unrelated negative-stranded viruses (NSV) expressing HIV-1 Gag as vaccine vectors: rabies virus (RABV), vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV). We hypothesized that this approach would result in more robust cellular immune responses than those achieved with the use of any of the vaccines alone in a homologous prime-boost regimen. To this end, we primed BALB/c mice with each of the NSV-based vectors. Primed mice were rested for thirty-five days after which we administered a second immunization with the same or heterologous NSV-Gag viruses. The magnitude and quality of the Gag-specific CD8⁺ T cells in response to these vectors post boost were measured. In addition, we performed challenge experiments using vaccinia virus expressing HIV-1 Gag (VV-Gag) thirty-three days after the boost inoculation. Our results showed that the choice of the vaccine used for priming was important for the detected Gag-specific CD8⁺ T cell recall responses post boost and that NDV-Gag appeared to result in a more robust recall of CD8⁺ T cell responses independent of the prime vaccine used. However, the different prime-boost strategies were not distinct for the parameters studied in the challenge experiments using VV-Gag but did indicate some benefits compared to single immunizations. Taken together, our data show that NSV vectors can individually stimulate HIV-Gag specific CD8⁺ T cells that are effectively recalled by other NSV vectors in a heterologous prime-boost approach. These results provide evidence that RABV, VSV and NDV can be used in combination to develop vaccines needing prime-boost regimens to stimulate effective immune responses.     

Pre-Clinical Development of BCG.HIVA(CAT), an Antibiotic-Free Selection Strain, for HIV-TB Pediatric Vaccine Vectored by Lysine Auxotroph of BCG

In the past, we proposed to develop a heterologous recombinant BCG prime-recombinant modified vaccinia virus Ankara (MVA) boost dual pediatric vaccine platform against transmission of breast milk HIV-1 and Mycobacterium tuberculosis (Mtb). In this study, we assembled an E. coli-mycobacterial shuttle plasmid pJH222.HIVA^CAT expressing HIV-1 clade A immunogen HIVA. This shuttle vector employs an antibiotic resistance-free mechanism based on Operator-Repressor Titration (ORT) system for plasmid selection and maintenance in E. coli and lysine complementation in mycobacteria. This shuttle plasmid was electroporated into parental lysine auxotroph (safer) strain of BCG to generate vaccine BCG.HIVA^CAT. All procedures complied with Good Laboratory Practices (GLPs). We demonstrated that the episomal plasmid pJH222.HIVA^CAT was stable in vivo over a 20-week period, and genetically and phenotypically characterized the BCG.HIVA^CAT vaccine strain. The BCG.HIVA^CAT vaccine in combination with MVA.HIVA induced HIV-1- and Mtb-specific interferon γ-producing T-cell responses in newborn and adult BALB/c mice. On the other hand, when adult mice were primed with BCG.HIVA^CAT and boosted with MVA.HIVA.85A, HIV-1-specific CD8⁺ T-cells producing IFN-γ, TNF-α, IL-2 and CD107a were induced. To assess the biosafety profile of BCG.HIVA^CAT-MVA.HIVA regimen, body mass loss of newborn mice was monitored regularly throughout the vaccination experiment and no difference was observed between the vaccinated and naïve groups of animals. Thus, we demonstrated T-cell immunogenicity of a novel, safer, GLP-compatible BCG-vectored vaccine using prototype immunogen HIVA. Second generation immunogens derived from HIV-1 as well as other major pediatric pathogens can be constructed in a similar fashion to prime protective responses soon after birth.     

Evaluation of Novel Human Immunodeficiency Virus Type 1 Gag DNA Vaccines for Protein Expression in Mammalian Cells and Induction of Immune Responses

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) are an important parameter of host defenses that limit viral replication after infection. Induction of effective CTL against conserved viral proteins such as Gag may be essential to the development of a safe and effective HIV type 1 (HIV-1) vaccine. DNA vaccination represents a novel strategy for inducing potent CD8(+) CTL responses in vivo. However, expression of HIV-1 structural proteins by DNA vectors has been hampered by a stringent requirement for coexpression with other viral components, such as Rev and RRE. Furthermore, even with Rev and RRE present, the level of expression of HIV-1 Gag, Pol, or Env is very low in murine cells. These problems have limited our ability to address the key issue of how to generate effective CTL responses to Gag in a mouse model. To overcome this problem, we compared several novel DNA expression vectors for HIV-1 Gag protein expression in primate and mouse cells and for generating immune responses in mice after DNA vaccination. A DNA vector containing wild type HIV-1 gag coding sequences did not induce detectable Gag expression in any of the cells tested. Attempts to increase nuclear export of Gag expression RNA by adding the constitutive transport element yielded only a moderate increase in Gag expression in monkey-derived COS cells and an even lower increase in Gag expression in HeLa cells or several mouse cell lines. In contrast, silent-site mutations in the HIV-1 gag coding sequences significantly increased Gag expression levels in all cells tested. Furthermore, this construct induced both Gag-specific antibody and CTL responses in mice after DNA vaccination. Using this construct, we achieved stable expression of HIV-1 Gag in the mouse cell line p815, which can now be used as a target cell for measuring HIV-1 Gag-specific CTL responses in immunized mice. The DNA vectors described in this study should make it possible to systematically evaluate the approaches for maximizing the induction of CTL responses against HIV-1 Gag in mouse and other animal systems.     

Mycobacterium tuberculosis Infection Interferes with HIV Vaccination in Mice

Tuberculosis (TB) has emerged as the most prominent bacterial disease found in human immunodeficiency virus (HIV)-positive individuals worldwide. Due to high prevalence of asymptomatic Mycobacterium tuberculosis (Mtb) infections, the future HIV vaccine in areas highly endemic for TB will often be administrated to individuals with an ongoing Mtb infection. The impact of concurrent Mtb infection on the immunogenicity of a HIV vaccine candidate, MultiHIV DNA/protein, was investigated in mice. We found that, depending on the vaccination route, mice infected with Mtb before the administration of the HIV vaccine showed impairment in both the magnitude and the quality of antibody and T cell responses to the vaccine components p24Gag and gp160Env. Mice infected with Mtb prior to intranasal HIV vaccination exhibited reduced p24Gag-specific serum IgG and IgA, and suppressed gp160Env-specific serum IgG as compared to respective titers in uninfected HIV-vaccinated controls. Importantly, in Mtb-infected mice that were HIV-vaccinated by the intramuscular route the virus neutralizing activity in serum was significantly decreased, relative to uninfected counterparts. In addition mice concurrently infected with Mtb had fewer p24Gag-specific IFN-γ-expressing T cells and multifunctional T cells in their spleens. These results suggest that Mtb infection might interfere with the outcome of prospective HIV vaccination in humans.     

Maternal LAMP/p55gagHIV-1 DNA immunization induces in utero priming and a long-lasting immune response in vaccinated neonates

Infants born to HIV-infected mothers are at high risk of becoming infected during gestation or the breastfeeding period. A search is thus warranted for vaccine formulations that will prevent mother-to-child HIV transmission. The LAMP/gag DNA chimeric vaccine encodes the HIV-1 p55gag fused to the lysosome-associated membrane protein-1 (LAMP-1) and has been shown to enhance anti-Gag antibody (Ab) and cellular immune responses in adult and neonatal mice; such a vaccine represents a new concept in antigen presentation. In this study, we evaluated the effect of LAMP/gag DNA immunization on neonates either before conception or during pregnancy. LAMP/gag immunization of BALB/c mice before conception by the intradermal route led to the transfer of anti-Gag IgG1 Ab through the placenta and via breastfeeding. Furthermore, there were an increased percentage of CD4+CD25+Foxp3+T cells in the spleens of neonates. When offspring were immunized with LAMP/gag DNA, the anti-Gag Ab response and the Gag-specific IFN-γ-secreting cells were decreased. Inhibition of anti-Gag Ab production and cellular responses were not observed six months after immunization, indicating that maternal immunization did not interfere with the long-lasting memory response in offspring. Injection of purified IgG in conjunction with LAMP/gag DNA immunization decreased humoral and cytotoxic T-cell responses. LAMP/gag DNA immunization by intradermal injection prior to conception promoted the transfer of Ab, leading to a diminished response to Gag without interfering with the development of anti-Gag T- and B-cell memory. Finally, we assessed responses after one intravenous injection of LAMP/gag DNA during the last five days of pregnancy. The intravenous injection led to in utero immunization. In conclusion, DNA vaccine enconding LAMP-1 with Gag and other HIV-1 antigens should be considered in the development of a protective vaccine for the maternal/fetal and newborn periods.