Impact of MHC class I alleles on the M. tuberculosis antigen-specific CD8+ T-cell response in patients with pulmonary tuberculosis.

Challenged by scattered understanding of protective immunity to Mycobacterium tuberculosis (MTB), we have mapped peptide epitopes to human leukocyte antigen (HLA)-A*0101, A*0201, A*1101, A*2402, B*0702, B*0801 and B*1501 of the secreted mycobacterial antigen Ag85B, a vaccine candidate that may be associated with immune protection. Affinity (ED(50)) and half-life (t(1/2), off-rate) analysis for individual peptide species on HLA-A and HLA-B molecules revealed binding ranges between 10(-3) and 10(-7) M. After selection of the best matches, major histocompatibility complex class I/peptide tetramer complexes were constructed to measure the CD8+ T-cell responses directly ex vivo in peripheral blood mononuclear cells (PBMC) derived from 57 patients with acute pulmonary tuberculosis. Three patterns of (allele-) specific CD8+ recognition were identified: (a). Focus on one dominant epitope with additional recognition of several subdominant T-cell epitopes (HLA-A*0301, A*2402, B*0801 and B*1501); (b). Co-dominant recognition of two distinct groups of peptides presented by HLA-B*0702; and (c). Diverse and broad recognition of peptides presented by HLA-A*0201. Peptides that bound with slow off-rates to class I alleles, that is HLA-A*0201, were associated with low frequency of CD8+ T cells in PBMCs from patients with tuberculosis. HLA-B alleles showed fast off-rates in peptide binding and restricted high numbers (up to 6%) of antigen-specific CD8+ T cells in patients with pulmonary tuberculosis.

Progress toward the development of a bacterial vaccine vector that induces high-titer long-lived broadly neutralizing antibodies against HIV-1.

Conformationally constrained HIV-1 Env and gp120 immunogens induce broadly cross-reactive neutralizing antibodies. Thus, it is now feasible to rationally design an HIV-1 vaccine that affords protection through humoral mechanisms. This paper reviews our progress toward the development of an oral bacterial vaccine vector that is capable of delivering an HIV-1 DNA vaccine to host lymphoid tissues and inducing broadly neutralizing antibodies to HIV-1 in the mucosal and systemic immune compartments.

Immunogenicity of DNA vaccines that direct the coincident expression of the 120 kDa glycoprotein of human immunodeficiency virus and the catalytic domain of cholera toxin.

Passive antibody studies unequivocally demonstrate that sterilizing immunity against lentiviruses is obtainable through humoral mechanisms. In this regard, DNA vaccines represent an inexpensive alternative to subunit vaccine for mass vaccination programs designed to induce such responses to human immunodeficiency virus type I (HIV-1). At present, however, this vaccine modality has proven relatively ineffective at inducing humoral responses. In this report, we describe the immunogenicity of DNA vaccines that direct the coincident expression of the cholera toxin catalytic domain (CTA1) with that of the human immunodeficiency virus type I gp120 through genes either encoded in individual plasmids or in a single dicistronic plasmid. In BALB/cJ mice, coincident expression of CTA1 in either a separate plasmid or in the dicistronic plasmid in the DNA vaccines induced serum IgG responses to gp120 that were at least 1000-fold greater, and remained elevated longer than, the analogous responses in mice vaccinated with a DNA vaccine that expressed gp120 alone. In addition, mice vaccinated with CTA1 and gp120 produced significantly more gp120-specific IFN-gamma ELISPOTs than mice vaccinated with the gp120 DNA vaccine. Combined, these data show that the adjuvant properties of cholera toxin can be harnessed in DNA vaccine modalities.

A Tat subunit vaccine confers protective immunity against the immune-modulating activity of the human immunodeficiency virus type-1 Tat protein in mice.

The rational design of new therapies against HIV-1 necessitates an improved understanding of the mechanisms underlying the production of ineffective immune responses to HIV-1 in most infected individuals. This report shows that the CD8(+) T cell responses to gp120 were greatly diminished in mice vaccinated with a bicistronic gp120-Tat DNA vaccine, compared with those induced by a DNA vaccine encoding gp120 alone. The CD8(+) T cell responses induced by the latter included strong gp120-specific IFN-gamma secretion and protective antiviral immunity against challenge by a vaccinia-env pseudotype. The degree to which Tat influenced CD8(+) T cell responses depended on the bioactivity of Tat. Thus, a bicistronic DNA vaccine that expresses gp120 and a truncated Tat defective for LTR activation elicited strong IFN-gamma -secreting CD8(+) T cell responses to gp120 but conferred only marginal protection against the vaccinia-env challenge. The effect of Tat was completely blocked, however, by immunization with inactivated Tat protein before vaccination with the bicistronic gp120-Tat DNA vaccine.

Mucosal and systemic HIV-1 Env-specific CD8(+) T-cells develop after intragastric vaccination with a Salmonella Env DNA vaccine vector.

CD8(+) T-cell responses provide beneficial antiviral immunity against human immunodeficiency virus 1 (HIV-1). In this study, we show that intragastric vaccination with a Salmonella HIV-1 Env DNA vaccine vector generates Env-specific CD8(+) T-cells, both in mucosal and systemic lymphoid tissue. By contrast, intramuscular vaccination with the Env DNA vaccine alone only induced systemic CD8(+) T-cells. To our knowledge, this is the first report showing both mucosal and systemic CD8(+) T-cell responses following vaccination with a Salmonella vaccine vector. These data suggest that this mode of HIV-1 DNA vaccine delivery will be advantageous over parenterally administered HIV-1 DNA vaccines.

Vaccination with a Shigella DNA vaccine vector induces antigen-specific CD8(+) T cells and antiviral protective immunity.

A prototype Shigella human immunodeficiency virus type 1 (HIV-1) gp120 DNA vaccine vector was constructed and evaluated for immunogenicity in a murine model. For comparative purposes, mice were also vaccinated with a vaccinia virus-env (vaccinia-env) vector or the gp120 DNA vaccine alone. Enumeration of the CD8(+)-T-cell responses to gp120 after vaccination using a gamma interferon enzyme-linked spot assay revealed that a single intranasal dose of the Shigella HIV-1 gp120 DNA vaccine vector elicited a CD8(+) T-cell response to gp120, the magnitude of which was comparable to the sizes of the analogous responses to gp120 that developed in mice vaccinated intraperitoneally with the vaccinia-env vector or intramuscularly with the gp120 DNA vaccine. In addition, a single dose of the Shigella gp120 DNA vaccine vector afforded significant protection against a vaccinia-env challenge. Moreover, the number of vaccinia-env PFU recovered in mice vaccinated intranasally with the Shigella vector was about fivefold less than the number recovered from mice vaccinated intramuscularly with the gp120 DNA vaccine. Since the Shigella vector did not express detectable levels of gp120, this report confirms that Shigella vectors are capable of delivering passenger DNA vaccines to host cells and inducing robust CD8(+) T-cell responses to antigens expressed by the DNA vaccines. Furthermore, to our knowledge, this is the first documentation of antiviral protective immunity following vaccination with a live Shigella DNA vaccine vector.

Recent advances with recombinant bacterial vaccine vectors.

Bacille Calmette-Guerin (BCG), Listeria monocytogenes, Salmonellae and Shigellae have shown promise as vaccine vectors in experimental animal models. Although disappointing results in humans and non-human primates stalled the development of this vaccination strategy, interest in this approach was reinvigorated recently by the development of bacterial DNA-vaccine-vectors. The purpose of this review is to highlight the strengths and weaknesses of bacterial vaccine vectors, and to discuss the future prospects of these vaccine delivery systems.

Expression of recombinant enterotoxigenic Escherichia coli colonization factor antigen I by Salmonella typhimurium elicits a biphasic T helper cell response.

Protective immunity to enterotoxigenic Escherichia coli (ETEC) is antibody (Ab) dependent; however, oral immunization with purified ETEC fimbriae fails to elicit protective immunity as a consequence of antigenic alteration by the gastrointestinal (GI) tract. Unless unaltered ETEC fimbriae can reach the inductive lymphoid tissues of the GI tract, immunity to ETEC cannot be induced. To produce immunity, live vectors, such as Salmonella typhimurium, can effectively target passenger antigens to the inductive lymphoid tissues of the GI tract. By convention, oral immunizations with Salmonella vectors induce CD4(+) T helper (Th) cell responses by gamma interferon (IFN-gamma)-dominated pathways both to the vector and passenger antigen, resulting in serum immunoglobulin G2a (IgG2a) and modest mucosal IgA Ab responses. In the present study, mice orally immunized with a Salmonella vector engineered to stably express ETEC colonization factor antigen I (CFA/I) showed initially elevated serum IgG1 and mucosal IgA anti-CFA/I Ab responses. As expected, mice orally immunized with an E. coli-CFA/I construct elicited poor anti-CFA/I Ab responses. In fact, the addition of cholera toxin during oral E. coli-CFA/I immunization failed to greatly enhance mucosal IgA Ab responses. Seven days after immunization with the Salmonella-CFA/I construct, cytokine-specific ELISPOT showed induction of predominant Th2-type responses in both mucosal and systemic immune compartments supporting the early IgG1 and IgA anti-CFA/I Abs. By 4 weeks, the Th cell response became Th1 cell dominant from the earlier Th2-type responses, as evidenced by increased mucosal and systemic IFN-gamma-producing T cells and a concomitant elevation of serum IgG2a Ab responses. This biphasic response offers an alternative strategy for directing Salmonella vector-induced host immunity along a Th2 cell-dependent pathway, allowing for early promotion of mucosal and systemic Abs.

Regulation of host immune responses by modification of Salmonella virulence genes.

Modifying bacterial virulence genes to probe the nature of host immunity is mostly unexplored. Here we investigate whether host immune responses can be regulated by modification of bacterial virulence genes. In mice, attenuated Salmonella mutant strains with clinical relevance elicited differential host immune responses. Oral administration of a mutant strain with a PhoP-null phenotype promoted potent innate immune responses of macrophages that were sufficient for host defense. In contrast, administration of an Aro- mutant strain elicited stronger specific antibody and T-helper (Th)-cell responses, wherein Th1-type cells were required for clearance. Thus, genetic manipulation of bacteria may be used to broadly alter immune mechanisms that regulate attenuation within the host and to tailor host immunity to specific bacterial pathogens.

Oral immunization with a Salmonella typhimurium vaccine vector expressing recombinant enterotoxigenic Escherichia coli K99 fimbriae elicits elevated antibody titers for protective immunity.

Bovine enterotoxigenic Escherichia coli (ETEC) continues to cause mortality in piglets and newborn calves. In an effort to develop a safe and effective vaccine for the prevention of F5(+) ETEC infections, a balanced lethal asd+ plasmid carrying the complete K99 operon was constructed and designated pMAK99-asd+. Introduction of this plasmid into an attenuated Salmonella typhimurium Deltaaro Deltaasd strain, H683, resulted in strain AP112, which stably expresses E. coli K99 fimbriae. A single oral immunization of BALB/c and CD-1 mice with strain AP112 elicited significant mucosal immunoglobulin A (IgA) titers that remained elevated for >11 weeks. IgA and IgG responses in serum specific for K99 fimbriae were also induced, with a prominent IgG1, as well as IgG2a and IgG2b, titer. To assess the derivation of these antibodies, a K99 isotype-specific B-cell ELISPOT analysis was conducted by using mononuclear cells from the lamina propria of the small intestines (LP), Peyer's patches (PP), and spleens of vaccinated and control BALB/c mice. This analysis revealed elevated numbers of K99 fimbria-specific IgA-producing cells in the LP, PP, and spleen, whereas elevated K99 fimbria-specific IgG-producing cells were detected only in the PP and spleen. These antibodies were important for protective immunity. One-day-old neonates from dams orally immunized with AP112 were provided passive protection against oral challenge with wild-type ETEC, in contrast to challenged neonates from unvaccinated dams or from dams vaccinated with a control Salmonella vector. These results confirm that oral Salmonella vaccine vectors effectively deliver K99 fimbriae to mucosal inductive sites for sustained elevation of IgA and IgG antibodies and for eliciting protective immunity.

Differential early interactions between Salmonella enterica serovar Typhi and two other pathogenic Salmonella serovars with intestinal epithelial cells.

Salmonella enterica serovar Typhi (hereafter referred to as S. typhi) is a host-restricted pathogen that adheres to and invades the distal ileum and subsequently disseminates to cause typhoid fever in humans. However, S. typhi appears to be avirulent in small animals. In contrast, other pathogenic salmonellae, such as S. enterica serovars Typhimurium and Dublin (S. typhimurium and S. dublin, respectively), typically cause localized gastroenteritis in humans but have been used as models for typhoid fever because these organisms cause a disease in susceptible rodents that resembles human typhoid. In vivo, S. typhi has been demonstrated to attach to and invade murine M cells but is rapidly cleared from the Peyer's patches without destruction of the M cells. In contrast, invasion of M cells by S. typhimurium is accompanied by destruction of these M cells and subsequently sloughing of the epithelium. These data have furthered our view that the early steps in the pathogenesis of typhoidal and nontyphoidal Salmonella serovars are distinct. To extend this concept, we have utilized an in vitro model to evaluate three parameters of initial host-pathogen interactions: adherence of three Salmonella serovars to human and murine small intestinal epithelial cell (IEC) lines, the capacity of these salmonellae to invade IECs, and the ability of the bacteria to induce interleukin-6 (IL-6) in these cell lines as a measure of host cell activation and the host acute-phase response. The results demonstrate that S. typhi adheres to and invades human small IECs better than either S. typhimurium or S. dublin. Interestingly, invA and invE null mutants of S. typhi are able neither to adhere to nor to invade IECs, unlike S. typhimurium invA and invE mutants, which adhere to but cannot invade IECs. S. typhi also induces significantly greater quantities of IL-6 in human small IEC lines than either of the other two Salmonella serovars. These findings suggest that differential host cytokine responses to bacterial pathogens may play an important role in the pathological sequelae that follow infection. Importantly, S. typhimurium did not induce IL-6 in murine IECs. Since S. typhimurium infection in mice is often used as a model of typhoid fever, these findings suggest that, at least in this case, the mouse model does not reflect the human disease. Taken together, our studies indicate that (i) marked differences occur in the initial steps of S. typhi, S. typhimurium, and S. dublin pathogenesis, and (ii) conclusions about S. typhi pathogenesis that have been drawn from the mouse model of typhoid fever should be interpreted conservatively.

Lipopolysaccharide from an Escherichia coli htrB msbB mutant induces high levels of MIP-1 alpha and MIP-1 beta secretion without inducing TNF-alpha and IL-1 beta.

OBJECTIVE: To identify a lipopolysaccharide (LPS) that retains the capacity to induce beta-chemokine secretion without the concomitant activation of pyrogenic cytokines. METHODS: LPS was extracted from strain MLK986 (mLPS), an htrB1::Tn10, msbB::ocam mutant of Escherichia coli that is defective for lipid A synthesis, and from wild-type parent E coli strains, W3110 (wtLPS). The capacity of these LPS preparations to induce tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and macrophage inflammatory proteins 1 alpha (MIP-1 alpha) and MIP-1 beta was assessed using a human peripheral blood mononuclear cell (PBMC) activation assay. RESULTS: Stimulation of PBMCs with mLPS did not induce measurable levels of pyrogenic cytokines TNF-alpha and IL-1 beta, whereas wtLPS induced high levels of these cytokines. Furthermore, mLPS antagonized the induction of TNF-alpha secretion by wtLPS. Nonetheless, mLPS retained a discrete agonist activity that induced MIP-1 alpha and MIP-1 beta secretion by PBMCs. This latter agonist activity appears to be unique to mLPS, since two previously documented LPS antagonists, Rhodobacter sphaeroides diphosphoryl lipid A and synthetic lipid IVA, did not induce MIP-1 alpha and MIP-1 beta secretion. Furthermore, synthetic lipid IVA was an antagonist of MIP-1 alpha and MIP-1 beta induction by mLPS. CONCLUSION: These results show that mLPS exhibits a novel bipartite activity, being an effective antagonist of TNF-alpha induction by wtLPS, while paradoxically being an agonist of MIP-1 alpha and MIP-1 beta secretion.

Induction of mucosal and systemic responses against human immunodeficiency virus type 1 glycoprotein 120 in mice after oral immunization with a single dose of a Salmonella-HIV vector.

Previous studies from our group showed that a Salmonella-HIV vector vaccine that expressed recombinant HIV-1 envelope protein gp120 stably in the vector cytoplasm elicited type 1 helper T cell (Th1) responses to gp120. Despite the promise of such vaccines, a major limitation in their use was that multiple immunizations were required to elicit even small responses. For this reason, we sought a modified vector configuration that would induce more potent gp120-specific T cell responses exhibiting a broader spectrum of effector functions after a single inoculation. In this article we describe the construction and immunogenicity of a Salmonella-HIV vector that displays a truncated derivative of HIV-1(IIIB) envelope in the periplasm of the vector. A single oral dose of this Salmonella vector, called H683(pW58-asd+), generated a gp120-specific proliferation response in the spleen 14 days after immunization. In agreement with our previous findings, the gp120-specific splenic CD4+ T cells elicited by H683(pW58-asd+) displayed a Th1 phenotype; however, gp120-specific splenic CD4+ Th2 cells were also evident. In addition, this strain induced strong gp120-specific IgA antibody-secreting cell (ASC) responses in the intestinal lamina propria and mesenteric lymph nodes. As many as 2% of the total lamina propria and mesenteric lymph node IgA ASCs were found to be specific for gp120 28 days after a single oral dose of H683(pW57-asd+). Because the proliferative response following a single dose of H683(pW58-asd+) was comparable to that seen previously after three doses of an analogous construct expressing recombinant gp120 in the cytoplasm, these observations suggest that Salmonella-vectored secreted HIV-1 antigens elicit higher T cell responses than their cytoplasmically bound analogs.

Release of tumor necrosis factor alpha in response to Vibrio vulnificus capsular polysaccharide in in vivo and in vitro models.

Vibrio vulnificus produces a severe septic shock syndrome in susceptible individuals. Virulence of the bacterium has been closely linked to the presence of a surface-exposed acidic capsular polysaccharide (CPS). To investigate whether CPS plays an additional role in pathogenesis by modulating inflammatory-associated cytokine production, studies were initiated in a mouse model and followed by investigations of cytokine release from human peripheral blood mononuclear cells (PBMCs). Mouse tumor necrosis factor alpha (TNF-alpha) could be detected in serum up to 12 h postinoculation in animals challenged with the encapsulated parent strain MO6-24/O. The unencapsulated strain CVD752 was quickly eliminated by the animals, thus preventing a direct association between serum TNF-alpha levels and the presence or absence of the CPS. Purified CPS from MO6-24/O when injected into D-galactosamine-sensitized mice was a more immediate inducer of TNF-alpha than an equivalent quantity of MO6-24/O lipopolysaccharide (LPS). Both V. vulnificus CPS and V. vulnificus LPS induced inflammation-associated cytokine responses from primary human PBMCs in vitro. CPS elicited TNF-alpha from PBMCs in a dose-dependent manner, with maximal induction at 6 to 10 h, and was not inhibited by polymyxin B. Expression of interleukin-6 (IL-6) mRNAs was also induced in the presence of CPS. Interestingly, while adherent PBMCs secreted high levels of TNF-alpha after stimulation with LPS, they secreted little TNF-alpha in response to CPS. These studies provide evidence that V. vulnificus CPS directly stimulates the expression and secretion of proinflammatory cytokines by murine and human cells and suggest that CPS activation of PBMCs operates through a cellular mechanism distinct from that of LPS.

Oral bacterial vaccine vectors for the delivery of subunit and nucleic acid vaccines to the organized lymphoid tissue of the intestine.

Bacterial vaccine vectors have the potential to deliver a number of antigens from bacterial, protozoan and viral pathogens. To further develop the utility of bacterial vaccine vectors we are currently evaluating three model systems: 1. A Salmonella-ETEC Vaccine Vector; 2. A Salmonella-HIV Vaccine Vector, and 3. Novel Live Bacterial Nucleic Acid Vaccine Vectors. Through our studies, and those of others, significant progress has been made toward bacterial vaccine vector systems that effectively deliver subunit and nucleic acid vaccines to the organized lymphoid tissue of the intestine. The practical reality of these findings is discussed.

Optimization of live oral Salmonella-HIV-1 vaccine vectors for the induction of HIV-specific mucosal and systemic immune responses.

Recent evidence suggests that live oral Salmonella-HIV vaccine vectors have the potential to elicit HIV-specific T cell-mediated immunity in both the mucosal and systemic compartments. We are using the mouse-typhoid model to identify Salmonella::HIV vaccine vector constructs that elicit HIV-specific mucosal and systemic immune responses. Oral immunization of mice with a Salmonella strain that expresses recombinant gp120 (rgp120) in the cytoplasm of the vector elicits a modest gp120-specific T cell proliferation response in the spleen. However, such Salmonella constructs did not stimulate the development of gp120-specific serum IgG or cytotoxic T lymphocytes (CTLs). Interestingly, the majority of cytoplasmically-expressed rgp120 forms inclusion bodies in Salmonella. We believe that in this form rgp120 is highly susceptible to protease degradation by the vector. As such, cytoplasmic rgp120 may not persist in the host after vaccination, resulting in the modest immunogenicity of rgp120 in these constructs. To circumvent this problem we constructed Salmonella strains that express rgp120 on the surface of the vector. Preliminary data suggest that surface-expressed rgp120 is significantly more immunogenic in both the mucosal and systemic compartments than cytoplasmic rgp120. These results, therefore, support the proposal that Salmonella vectors will be a safe and inexpensive means for delivery of HIV antigens to, and the elicitation of HIV-specific T cells in, the mucosal and systemic compartments.

Immune responses to novel Escherichia coli and Salmonella typhimurium vectors that express colonization factor antigen I (CFA/I) of enterotoxigenic E. coli in the absence of the CFA/I positive regulator cfaR.

An asd-stabilized plasmid carrying enterotoxigenic Escherichia coli cfaABCE genes was constructed and called pJGX15C-asd+. Expression of colonization factor antigen I (CFA/I) by this plasmid occurs independently of the cfaABCE positive regulator cfaR in attenuated Salmonella delta aro delta asd strain H683 and nonpathogenic laboratory E. coli asd strain chi 6212. Oral immunization of mice with nonpathogenic E. coli chi 6212 (pJGX15C-asd+) does not elicit significant serum or mucosal responses against CFA/I. In contrast, oral immunization with a single dose of attenuated S. typhimurium H683(pJGX15C-asd+) elicits a 10(5)-fold increase in CFA/I-specific serum immunoglobulin G and significant elevation of CFA/I-specific immunoglobulin A-secreting B cells in the lamina propria, mesenteric lymph nodes, and spleen. Thus, only the Salmonella-CFA/I construct effectively delivered CFA/I to the inductive sites of the gut-associated and systemic lymphoid tissues.

Construction and immunogenicity of Salmonella typhimurium vaccine vectors that express HIV-1 gp120.

Since the human immunodeficiency virus (HIV-1) is transmitted either parenterally or sexually, both mucosal and systemic immune responses may be required to provide protective immunity. Attenuated Salmonella vectors expressing heterologous antigen can stimulate responses in both compartments. To evaluate the utility of Salmonella vectors as an HIV-1 vector vaccine, a gene expression cassette encoding recombinant HIV-1 gp120 (rgp120) was integrated into the hisOGD locus of Salmonella typhimurium aroA strain, SL3261 (SL3261::120). To test if increased antigen expression potentiates immunogenicity, strains were constructed that express rgp120 from a multicopy asd-stabilized plasmid (SL7207 pYA:120). Immunoblot analysis demonstrated that SL7207 pYA:120 expressed approximately 50-fold more rgp120 than SL3261::120. Oral immunization of BALB/c mice with these strains did not stimulate an env-specific CTL response or a significant rise in antigp120 antibody titer as compared to controls. However, splenic T cells from SL7207 pYA::120 immunized mice proliferated upon restimulation with gp120 in vitro while splenocytes from SL3261::120 immunized mice did not, gp120 restimulated splenic T cells from SL7207 pYA:120 immune mice also produced IFN-gamma but no IL-5. Two conclusions can be drawn from these results. First, high level expression of rgp120 in Salmonella vectors is necessary to stimulate a gp120-specific immune response in mice. Second, Salmonella::rgp120 stimulates a gp120-specific Th1 response in mice. This is the first report to describe the construction of a Salmonella::rgp120 vector vaccine that is immunogenic in mice.

Construction and characterization of a Salmonella typhi-based human immunodeficiency virus type 1 vector vaccine.

Since the human immunodeficiency virus type 1 (HIV-1) is transmitted either parenterally or sexually, both systemic and mucosal immune responses might be required to provide protective immunity. One option is to express HIV proteins in attenuated Salmonella vectors that elicit immune responses in both compartments. The first step to constructing such a strain was achieved by integrating a gene expression cassette encoding recombinant HIV-1 gp120 (rgp120) into the aroC locus of an attenuated vaccine strain of S. typhi. This rgp120 expression cassette utilizes the strong constitutive promoter, P1pp/lacUV5, and produces rgp120 to 0.05-01% of the total bacterial cell protein. Immunoblot analysis shows that the S. typhi strains containing the integrated cassette express a protein that is both recognized by anti-gp120 monoclonal antibodies (mAbs) and is the appropriate size for nonglycosylated full-length gp120 (52 kDa). Immunoblot analysis also demonstrates that the recombinant S. typhi strains express the rgp120 as monomers and multimers found predominantly in the insoluble fraction of the bacteria. Antigen-capture ELISA, using antibodies specific for continuous epitopes on gp120, revealed that the exposure of these epitopes on S. typhi-expressed rgp120 differs from exposure of these epitopes on baculovirus-expressed rgp120 that binds CD4. Epitopes in the first conserved region (109-113) and the third conserved/fourth variable regions (376-380, 382-384, 395-400) are more "surface-exposed", while one epitope in the third variable region (313-324) is more "buried" relative to the corresponding epitopes of baculovirus expressed gp120. Antibodies recognizing discontinuous epitopes of the CD4 binding domain do not react with the S. typhi expressed rgp120.(ABSTRACT TRUNCATED AT 250 WORDS)

Construction and characterization of attenuated delta aroA delta virG Shigella flexneri 2a strain CVD 1203, a prototype live oral vaccine.

We engineered an oral Shigella vaccine prototype that can invade intestinal epithelial cells but cannot undergo extensive intracellular replication or extend to adjacent epithelial cells. Strain CVD 1203, derived from wild-type Shigella flexneri 2a by introducing deletions in chromosomal aroA and invasion plasmid virG, was highly attenuated in the Sereny test. Two 10(9)-CFU orogastric doses (2 weeks apart) stimulated production of secretory immunoglobulin A antibodies to S. flexneri 2a and protected against conjunctival sac challenge with virulent S. flexneri 2a.