Adenoviral vectors based on adenovirus type 35 (rAd35) have the advantage of low natural vector immunity and induce strong, insert-specific T- and B-cell responses, making them prime-candidate vaccine carriers. However, severe vector-genome instability of E1-deleted rAd35 vectors was observed, hampering universal use. The instability of E1-deleted rAd35 vector proved to be caused by low pIX expression induced by removal of the pIX promoter, which was located in the E1B region of B-group viruses. Reinsertion of a minimal pIX promoter resulted in stable vectors able to harbour large DNA inserts (>5 kb). In addition, it is shown that replacement of the E4-Orf6 region of Ad35 by the E4-Orf6 region of Ad5 resulted in successful propagation of an E1-deleted rAd35 vector on existing E1-complementing cell lines, such as PER.C6 cells. The ability to produce these carriers on PER.C6 contributes significantly to the scale of manufacturing of rAd35-based vaccines. Next, a stable rAd35 vaccine was generated carrying Mycobacterium tuberculosis antigens Ag85A, Ag85B and TB10.4. The antigens were fused directly, resulting in expression of a single polyprotein. This vaccine induced dose-dependent CD4+ and CD8+ T-cell responses against multiple antigens in mice. It is concluded that the described improvements to the rAd35 vector contribute significantly to the further development of rAd35 carriers for mass-vaccination programmes for diseases such as tuberculosis, AIDS and malaria.

The HIV-1 Tat protein has been recently explored as a prospective vaccine candidate with broad, subtype non-specific action. We approached the problem of delivery of Tat through the mucosal route by expressing Tat in an edible plant. The tat gene was assembled from synthetic overlapping oligonucleotides, and was subsequently cloned into a plant virus-based vector tobacco mosaic virus (TMV). Spinach plants inoculated with the Tat-producing constructs were collected and fed to mice 7–14 days post inoculation. DNA vaccinations were performed using a gene gun. Codon optimization of the Tat gene expressed in spinach plants resulted in several-fold yield increase as detected in immunoblots, and did not cause severe symptoms in inoculated plants. Mice were fed with the Tat-producing or control vector-inoculated spinach. After three feedings, 1 week apart, 1 g per mice, no differences were detected in the growth rate or behavior of the animals fed with these three types of spinach. None of the animals developed measurable Tat antibodies. Following DNA vaccination, however, mice having previously received oral Tat developed higher antibody titers to Tat than did the controls, with the titers peaking at 4 weeks post-vaccination. Codon optimization allows production of up to 300–500 μg of Tat antigen per 1 g of leaf tissue in spinach using a plant virus-based expression system. The plant produced Tat does not seem to have any apparent adverse effect on mice growth or behavior, when fed with spinach for 4 weeks. ELISA data suggested that oral Tat primed for the development of Tat antibodies when mice were subsequently vaccinated with plasmid DNA designed for Tat expression.

Human herpesvirus 8 (HHV-8), the etiologic agent of Kaposi's sarcoma (KS), encodes a chemokine receptor homologue, the viral G protein-coupled receptor (vGPCR), that has been implicated in KS pathogenesis. Expression of vGPCR constitutively activates several signaling pathways, including NF-kappaB, and induces the expression of proinflammatory and angiogenic factors, consistent with the inflammatory hyperproliferative nature of KS lesions. Here we show that vGPCR also constitutively activates the nuclear factor of activated T cells (NF-AT), another transcription factor important in regulation of the expression of inflammatory cytokines and related factors. NF-AT activation by vGPCR depended upon signaling through the phosphatidylinositol 3-kinase-Akt-glycogen synthetase kinase 3 (PI3-K/Akt/GSK-3) pathway and resulted in increased expression of NF-AT-dependent cell surface molecules (CD25, CD29, Fas ligand), proinflammatory cytokines (interleukin-2 [IL-2], IL-4), and proangiogenic factors (granulocyte-macrophage colony-stimulating factor GMCSF and TNFalpha). vGPCR expression also increased endothelial cell-T-cell adhesion. Although infection with HHV-8 is necessary to cause KS, coinfection with human immunodeficiency virus type 1 (HIV-1), in the absence of antiretroviral suppressive therapy, increases the risk of KS by many orders of magnitude. NF-AT and NF-kappaB activation by vGPCR was greatly increased by the HIV-1 Tat protein, although Tat alone had little effect on NF-AT. The enhancement of NF-AT by Tat appears to be mediated through collaborative stimulation of the PI3-K/Akt/GSK-3 pathway by vGPCR and Tat. Our data further support the idea that vGPCR contributes to the pathogenesis of KS by a paracrine mechanism and, in addition, provide the first evidence of collaboration between an HIV-1 protein and an HHV-8 protein.

We conducted a national molecular epidemiologic survey of HIV-1 strains in Nigeria to determine the most prevalent subtype(s) for use in developing candidate vaccines. A total of 230 HIV-1-positive blood samples collected from 34 of the 36 Nigerian states were analyzed by our modified env gp41-based heteroduplex mobility assay (HMA) and/or gp41 sequencing and analysis. Overall, 103 (44.8%) were subtype A, 125 (54.3%) were subtype G, one (0.4%) was subtype C, and one (0.4%) was subtype J, and one (0.4%) was unclassifiable. To further characterize Nigerian viruses to aid in strain selection for candidate vaccines, one gp41 subtype G and five gp41 subtype A strains were selected for full envelope sequencing. The one subtype G sequence had consistent phylogenies throughout gp160, using programs to detect recombination. However, all five sequences that were primarily subtype A in gp41 were found to be recombinant viruses. Two of the five (40%) were A/G/J mosaics with common breakpoints. The remaining three gp160 recombinants all had their own unique break points: two A/? and one A/?/G, however, all five had the majority of their mosaic breakpoints occurring in gp41. None of the five were consistent with the circulating recombinant form (CRF)02_AG strain previously reported to be prevalent in West Africa. In conclusion, we showed a clear dominance and widespread distribution of gp41 subtypes A and G in fairly equal proportions, suggesting that vaccines designed for use in this geographic locale should incorporate the gene(s) of both subtypes. However, appreciating the magnitude of diversity of HIV-1 strains in Nigeria may require sequencing and analysis of longer gene regions for the identification of prevalent or emerging CRFs.

The gp120 region of the human immunodeficiency virus type 1 (HIV-1) envelope (env) gene exhibits a high level of genetic heterogeneity across the group M subtypes. The heteroduplex mobility assay (HMA) has successfully been used to assign subtype classifications, but C2V5 primers often fail to amplify African strains. We developed an env gp41-based HMA for which the target sequence is amplified with highly conserved gp41 primers, known to efficiently amplify nucleic acids from HIV-1 group M, N, and O viruses. By using gp41 from a new panel of reference strains, the subtype assignments made by our modified HMA were concordant with those obtained by sequencing and phylogenetic analysis of 34 field strains from 10 countries representing subtypes A to G. Testing of field strains from Nigeria further demonstrated the utility of this modified assay. Of 28 samples, all could be amplified with gp41 primers but only 17 (60.7%) could be amplified with the standard C2V5 primers. Therefore, gp41-based HMA can be a useful tool for the rapid monitoring of prevalent subtypes in countries with divergent strains of circulating HIV-1.

ABSTRACT The infection of CD4+ host cells by human immunodeficiency virus type 1 (HIV-1) is initiated by a temporal progression of interactions between specific cell surface receptors and the viral envelope protein, gp120. These interactions produce a number of intermediate structures with distinct conformational, functional, and antigenic features that may provide important targets for therapeutic and vaccination strategies against HIV infection. One such intermediate, the gp120-CD4 complex, arises from the interaction of gp120 with the CD4 receptor and enables interactions with specific coreceptors needed for viral entry. gp120-CD4 complexes are thus promising targets for anti-HIV vaccines and therapies. The development of such strategies would be greatly facilitated by a means to produce the gp120-CD4 complexes in a wide variety of contexts. Accordingly, we have developed single-chain polypeptide analogues that accurately replicate structural, functional, and antigenic features of the gp120-CD4 complex. One analogue (FLSC) consists of full-length HIV-1BaL gp120 and the D1D2 domains of CD4 joined by a 20-amino-acid linker. The second analogue (TcSC) contains a truncated form of the gp120 lacking portions of the C1, C5, V1, and V2 domains. Both molecules exhibited increased exposure of epitopes in the gp120 coreceptor-binding site but did not present epitopes of either gp120 or CD4 responsible for complex formation. Further, the FLSC and TcSC analogues bound specifically to CCR5 (R5) and blocked R5 virus infection. Thus, these single-chain chimeric molecules represent the first generation of soluble recombinant proteins that mimic the gp120-CD4 complex intermediate that arises during HIV replication.

Among the known colonization factors of enterotoxigenic Escherichia coli (ETEC), CFA/I and CS3 (the common antigen in the CFA/II family of fimbrial antigens) are two of the most prevalent fimbrial antigens found in clinical isolates but are never expressed by the same wild-type strain. We manipulated the genetic determinants encoding CS3 and CFA/I fimbriae so that these two important colonization factors are expressed simultaneously in attenuated Salmonella typhi live oral vaccine strain CVD 908, including after growth in liquid medium (CFA/I is poorly expressed by wild-type ETEC in broth culture). The recombinant fimbrial structures produced by CVD 908 are morphologically indistinguishable from the CS3 fibrillae and CFA/I rod-like fimbriae produced by ETEC, and are recognized by monospecific CS3 and CFA/I antibodies. This prototype construct may prove useful in investigating the live vector approach to immunoprophylaxis of ETEC diarrheal disease.

rcsp, encoding amino acids 21-398 of Plasmodium falciparum circumsporozoite protein (CSP), under control of tacP was integrated into the chromosomal delta aroC locus of attenuated delta aroC, delta aroD Salmonella typhi CVD 908. By immunoblot and ELISA, rCSP expression was greater from a multicopy plasmid than from the single chromosomal gene. CVD 908 omega (delta aroC1019::tacP-rcsp) was well tolerated by 10 volunteers who were fed two doses of 5 x 10(7) organisms 8 days apart. Seven subjects excreted the vaccine strain for 1-3 days. All subjects developed serologic responses to O and H antigens of the live vector, whereas 3 vaccinees responded to the foreign antigen: 1 developed an 80-fold rise in serum anti-sporozoite antibody, another had a 4-fold rise in antibody to a recombinant portion of CSP (residues 309-345), while a third vaccinee developed CSP-specific CD8+ cytotoxic T lymphocyte activity. This is the first report of attenuated S. typhi eliciting a human serologic or a cytotoxic T lymphocyte response to a foreign protein. Improved foreign gene expression should enhance immunogenicity.

The ompB operon, comprising the ompR and envZ genes, was cloned from a Salmonella typhi Ty2 cosmid bank and characterized by DNA sequence analysis. The S. typhi ompR and envZ genes contained open reading frames encoding proteins of 240 and 451 amino acids, respectively. Comparison with the Salmonella typhimurium OmpB protein sequences revealed 99.5% homology. The DNA sequence data were used to identify appropriate restriction sites for generating a defined deletion of 517 bp within the open reading frame of the ompR gene. This deletion was introduced by homologous recombination into the chromosomes of two S. typhi strains which already harbored defined deletions in both the aroC and aroD genes. The presence of the deletions within ompR was confirmed by Southern hybridization and sequencing of the DNA fragments surrounding the deleted regions by PCR. The S. typhi ompR mutants displayed a marked decrease in OmpC and OmpF porin expression as demonstrated by examination of outer membrane preparations. It was also found that S. typhi strains harboring the defined ompR deletions no longer agglutinated with Vi antiserum. However, when a functional ompB operon was introduced back into the S. typhi ompR mutants, either on a multicopy plasmid or as a single-copy chromosomal replacement, the Vi+ phenotype was restored. The levels of Vi synthesis were also found to be sensitive to different concentrations of sodium chloride present in the growth medium, although the levels of sensitivity varied between different isolates of S. typhi. It is therefore concluded that the ompR-envZ two component regulatory system plays an important role in the regulation of Vi polysaccharide synthesis in S. typhi and that one of the environmental signals for this regulation may be osmolarity.

New recombinant strains of attenuated Salmonella typhi used as live oral vaccines elicit potent immune responses. This study examined the patterns of cytokine production and proliferation to specific S. typhi antigens in subjects orally immunized with attenuated S. typhi vaccines CVD 906, CVD 908, and CVD 908 expressing the circumsporozoite protein of Plasmodium falciparum. After immunization, sensitized lymphocytes were found in subjects' blood that exhibited significantly increased proliferative responses and interferon-gamma production to purified S. typhi flagella when compared with preimmunization levels. Significant negative correlations were observed between interleukin-4 production and both interferon-gamma production and proliferation to S. typhi flagella. These results demonstrate that oral immunization with attenuated S. typhi strains alone or with those carrying a foreign gene elicits strong systemic cell-mediated immunity to purified S. typhi antigens, including the production of cytokines compatible with T1-type responses.

The construction of a Salmonella typhi Ty2 strain harbouring defined deletions in both the aroA and aroC genes is described. These deletions have been fully defined at the molecular level by DNA sequencing and have been introduced in such a way that no foreign DNA remains in the S. typhi genome. This strain is attenuated in mice when given by the intraperitoneal route suspended in hog gastric mucin and is attenuated to a similar level to strains harbouring deletions in aroA or aroC alone indicating that both lesions are capable of attenuating independently. We have used this defined S. typhi aroA aro C strain to express stably a non-toxic 50 kDa fragment of tetanus toxin (fragment C) from a gene incorporated into the chromosome. This strain has the advantage of harbouring no antibiotic-resistance markers and we consider it to be a candidate bivalent oral typhoid-tetanus vaccine.

The aroD gene from Salmonella typhi, encoding 5-dehydroquinate hydrolyase (3-dehydroquinase), has been cloned into Escherichia coli and the DNA sequence determined. The aroD gene was isolated from a cosmid gene bank by complementation of an S. typhimurium aroD mutant. Analysis of the DNA sequence revealed the presence of an open reading frame capable of encoding a protein of 252 amino acids with a calculated M(r) of 27706. Comparison of the deduced S. typhi 3-dehydroquinase protein sequence with that elucidated for E. coli revealed 69% homology. Alignment of the S. typhi sequence and equivalent Aspergillus nidulans and Saccharomyces cerevisiae sequences showed that homology was lower, at 24%, but still significant. Use of a minicell expression system demonstrated that a polyclonal antibody raised against E. coli 3-dehydroquinase cross-reacted with its S. typhi counterpart.

Oral immunization with an attenuated Salmonella typhimurium recombinant containing the full-length Plasmodium berghei circumsporozoite (CS) gene induces protective immunity against P. berghei sporozoite challenge in the absence of antibody. We found that this immunity was mediated through the induction of specific CD8+ T cells since in vivo elimination of CD8+ cells abrogated protection. In vitro studies revealed that this Salmonella-P. berghei CS recombinant induced class I-restricted CD8+ cytotoxic T cells that are directed against the P. berghei CS peptide epitope spanning amino acids 242-253. This is the same peptide that previously was identified as the target of cytotoxic T lymphocytes (CTL) induced by sporozoite immunization. Salmonella-P. falciparum CS recombinants were constructed that contained either the full-length CS gene or a repeatless gene consisting of CS flanking sequences. Both of these vaccines were able to induce CD8+ CTL directed against P. falciparum CS peptide 371-390, which is identical to the target of CTL induced by sporozoites and vaccinia CS recombinants. These results directly demonstrate the ability of an intracellular bacteria such as Salmonella to induce class I-restricted CD8+ CTL and illustrate the importance of CD8+ CTL in immunity to malaria.

In recent years there has been a resurgence of research to develop new and improved attenuated strains of Salmonella typhi to function as live oral vaccines against typhoid fever and to serve as "carrier" vaccines to express foreign antigens of other pathogens and deliver them to the immune system. Strain Ty21a has served as a prototype in clinical and field trials to identify the optimal formulations and dosage schedules for live vaccines and to quantitate the duration of protection that can be achieved. Clinical trials with three new attenuated S. typhi candidate vaccines, a Vi+ variant of Ty21a, an aroC, aroD double mutant recombinant strain and a cya, crp double mutant, are underway or will be initiated shortly.

We review evidence that the systemic and mucosal immune systems of the body are compartmentalized. The development of immunity against an antigen at one mucosal surface may lead to the appearance of that immunity at other mucosal surfaces. In order to attain good protective immunity against a bacterial enteropathogen, it may be necessary to induce such immunity through the mucosal immune system of the gut. Earlier attempts to elicit protective immunity against bacterial enteropathogens by parenteral vaccination are reviewed. The modern approach involves oral administration of antigen. Such antigen may consist of killed bacteria or--more effectively--live, attenuated bacteria bearing antigens of interest. Such bacteria may be enteropathogens attenuated by mutation, either general or site-directed, or hybrid strains in which a bacterial carrier expresses an antigenic determinant of interest from cloned DNA. While good progress has been made in the comprehension of the requirements for effective vaccination against enteropathogenic bacteria, future work will produce more effective carrier strains than are currently available.

We have developed a system whereby heterologous DNA encoding an antigen from an enteropathogen may be recombined into the chromosome of an attenuated Salmonella carrier strain. The system involves two steps; (i) integration of a hisOG deletion mutation into the chromosome; (ii) replacement of the hisOG deletion by the complete hisOG region and the segment of heterologous DNA which encodes the antigen of interest. Recombinants may be selected (his+). The system was used to integrate the genes encoding K88 fimbriae from enterotoxigenic Escherichia coli into the chromosome of a galE mutant of Salmonella typhimurium (LT2H1). Recombinants were detected at a frequency of between 1.0 × 10−3 and 1.5 × 10−3. A variety of tests confirmed that the K88 genes were integrated into the chromosome of LT2H1 and were expressed. The stability of the recombinant was tested both in vivo and in vitro. When administered orally to mice, the recombinant elicited a serum antibody response to K88, and retained the Salmonella vaccine potential of the vector strain.