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Keywords:

  • combination vaccine;
  • Chlamydia;
  • HSV-2;
  • antibodies;
  • cytokines;
  • protection

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Chlamydia trachomatis and Herpes simplex virus type 2 (HSV-2) genital infections pose a considerable public health challenge worldwide. Considering the high incidence of coinfections by the two pathogens, a combination vaccine that can be administered as a single regimen would be highly desirable. Recombinant Vibrio cholerae ghosts (rVCG) offer an attractive approach for the induction of humoral and cellular immune responses against human and animal pathogens. In this study, we evaluated a bivalent combination vaccine formulation comprising rVCG expressing chlamydial MOMP and HSV-2 glycoprotein D in mice for immunogenicity and protective efficacy against genital challenge with either pathogen. Mice immunized with the combination vaccine elicited secretory IgA and IgG2a antibodies to both chlamydial and HSV-2 antigens in serum and vaginal secretions. Robust antigen-specific mucosal and systemic T helper type 1 responses were induced in mice as measured by increased interferon-γ levels produced by immune T cells in response to restimulation with target antigen in vitro. In addition, mice immunized with the combination vaccine were prophylactically protected from genital challenge with high doses of live Chlamydia and HSV-2. Thus, the combination vaccine regimen delivered by rVCG elicited adequate immune effectors that simultaneously protected against the individual pathogens.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Sexually transmitted diseases (STDs) constitute a major reproductive health burden for sexually active individuals, disproportionately affecting the young, women, and communities comprising the poor and minorities (Aral & Holmes, 1999). An estimated 15 million new cases of STDs occur annually in the USA alone (Aral, 2001). The major complications of STDs include genital ulcers, pelvic inflammatory disease and related sequelae, ectopic pregnancy, infertility, and adverse outcomes of pregnancy (Aral, 2001). Genital infections caused by Chlamydia trachomatis and Herpes simplex virus type 2 (HSV-2) rank among the highest STDs in the world.

Most chlamydial genital tract infections in women are asymptomatic, with severe complications often being the first symptoms of an infection (Paavonen & Wolner-Hanssen, 1989; Schachter & Grayston, 1998). Although antibiotic therapy can effectively eliminate chlamydial infection, it does not always affect established pathology nor prevent reinfection (Stagg, 1998), and the rampant asymptomatic infections make treatment of symptomatic individuals alone unlikely to be a successful control strategy. The development and administration of a vaccine capable of protecting against infection or even ameliorating severe disease remains the most promising and effective strategy for controlling chlamydial infections (Stagg, 1998; Igietseme et al., 2002, 2003). The development of vaccines based on chlamydial subunit components is the current focus of chlamydial vaccine design. The major outer membrane protein (MOMP) is one of the leading subunit vaccine candidates. This 40-kDa immunodominant protein has been well characterized as a porin and an adhesin, and is a key determinant of chlamydial genus and species specificity.

Genital herpes is a widespread sexually transmitted disease that contributes significantly to morbidity and mortality in humans (Armstrong, 2001), with infections being particularly severe in neonates and immunocompromised individuals (Greenblatt, 1988; Whitley, 1991). In women, HSV-2 infects the mucosa in the genital tract and spreads to the nervous system. After the initial infection is resolved, latent virus can persist in infected ganglia for long periods, causing recurrent disease (Corey, 1994). Apart from the pain and discomfort associated with these infections, HSV-2 causes psychological trauma and may increase the risk of acquisition and transmission of HIV (Gwanzura, 1998; Chen, 2000). It can also be transmitted to fetuses or newborn infants, leading to spontaneous abortion or mental retardation (Whitley, 1991; Brown, 1997). Although antiviral drug therapy can reduce the severity of infections (Stanberry, 1999; De Clercq, 2000), some of the HSV strains have now developed resistance to these drugs (Naesens & De Clercq, 2001). Thus, the development of a vaccine to control genital herpes would greatly contribute to preventive health care (Bourne, 2003; Parr & Parr, 2003). Current challenges facing the development of an effective herpes vaccine include (i) the identification of an ideal immunogenic antigen, (ii) the delineation of the exact immune correlates of protection, and (iii) the development of an effective and safe immunization strategy (Grammer, 1990; Ghiasi, 1994; Blaney, 1998). Much of the research evaluating HSV subunit vaccines has centred on the use of surface glycoproteins as immunogens. The HSV glycoproteins B and D (gB and gD) are attractive choices for subunit vaccines because they are targets for both humoral- and cell-mediated immunity (class I and II restricted) (Burke, 1992). Both proteins also have a high degree of homology between HSV-1 and HSV-2, and, therefore, may provide protection against both types. Subunit vaccines containing gD or a combination of gB and gD have been shown to be immunogenic in animals and humans (Corey, 1999; Mester, 2000; Aurelian, 2004). In a recent clinical trial, immunization with gD recombinant protein mixed with alum and 3-O-deacylated-monophosphoryl lipid A adjuvants induced significant protection against clinically apparent genital herpes in women who were seronegative for both HSV-1 and HSV-2, although it failed to protect seropositive individuals (Stanberry, 2002).

Despite considerable effort and significant progress, no effective vaccine against either Chlamydia or genital herpes infection has yet been licensed. We have designed a novel recombinant Vibrio cholerae ghost (rVCG) delivery system, which has inherent adjuvant properties and is capable of simultaneously delivering multiple antigens from the same or different pathogens to the immune system (Eko, 2003, 2004). In this study, we constructed rVCG vector-based subunit vaccines expressing the chlamydial MOMP (rVCG-MOMP) or HSV-2 gD2 (rVCG-gD2). We then evaluated the immunogenicity and protective efficacy of a bivalent combination vaccine formulation comprising rVCG-MOMP and rVCG-gD2 in mice against genital challenge with live Chlamydia or HSV-2.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Chlamydia and HSV-2 stocks and antigen

Stock preparations of C. trachomatis serovar D strain were generated by propagating elementary bodies (EBs) in HeLa cells as previously described (Ramsey et al., 1988). All stocks were titrated on HeLa cell monolayers followed by purification of EBs over renografin gradients (Ramsey et al., 1988), and stored at −70°C. Herpes simplex virus type 2 (HSV-2 strain SR333) was propagated in Vero cells maintained on minimal essential medium (MEM) with 5% fetal calf serum (FCS) (Life Technologies, Gaithersburg, MD) and stored in aliquots at −80°C until used. Titers were measured in Vero cells and expressed as PFU mL−1. Chlamydial or HSV-2 antigen (UV-inactivated) was made by exposing chlamydial elementary bodies or the live virus to a Philips 30-W UV bulb for 30 min at a distance of 5 cm. HSV inactivation was confirmed by the inability to produce plaques when cultured on Vero cells.

Genomic DNA preparation and PCR amplification

Genomic DNA was purified from 1 × 108 chlamydial EBs or 1 × 106 PFU of free HSV-2 (strain SR 333) viral particles suspended in culture media using the QIAGEN DNeasy Tissue Kit (Qiagen, Valencia, CA) and QIAGEN's QIAamp DNA mini kit, respectively. Each protocol was carried out according to the manufacturer's instructions. The full-length omp1 and gD2 coding sequences were amplified without the leader peptides from purified genomic DNA using the Expand High Fidelity PCR System (Roche, Mannheim, Germany) and oligonucleotide primers flanked with specific restriction sites. The primer design was based on chlamydial and HSV-2 sequences obtained from published data (Hodgman & Minson, 1986; Stothard et al., 1998; Dolan, 1999; Kalman, 1999). The forward primer (EF-8F) for omp1 amplification incorporated an Apa1 restriction enzyme site with the sequence 5′-ggccgggcccagatgaaaaaactct-3′, and the reverse primer (EF-10R) incorporated a DraII site, 5′-gcgctagggcctattagaagcggaa-3′. For gD2 amplification, the forward primer (EF-43F) incorporated an XbaI restriction enzyme site (5′-cgtgtctagagaaatacgccttagc-3′), while the reverse primer (EF-44R) contained a HindIII site (5′-cgtcaagcttgctagtaaaacaatg-3′). The amplification reaction was carried out in an Eppendorf Gradient Mastercycler (Eppendorf, Hamburg, Germany), and the amplified PCR products were isolated from a 1% agarose gel and purified with the QlAquick PCR purification kit (Qiagen).

Construction of plasmids, pEL-gD2 and pCOM2

The gD2 expression vector was constructed by inserting the amplified gD2 PCR product (1107 bp) containing the full-length gD2 coding sequence, without the signal peptide, between the E' and L′ genes of vector pKSEL5-2 (Szostak & Lubitz, 1991), following restriction with Xba1 and HindIII endonucleases. T4 DNA ligase-mediated ligation was carried out, and the mixture was transformed into Escherichia coli DH5α. The resultant plasmid was designated as pEL-gD2. Construction of plasmid pCOM2, which harbors the full-length omp1 coding sequence, has been described previously (Eko, 2004). Briefly, the amplified omp1 PCR product (1207 bp) was inserted between the E′ and L′ genes of vector pKSEL5-2 at the SalI and PstI restriction sites following ligation with T4 DNA ligase (Roche), and transformed into E. coli DH5α. The resultant expression plasmid was designated as pCOM2. All constructs were analysed by restriction endonuclease digestion and sequencing of the junctions and coding regions of gD2 and omp1 genes.

Detection of recombinant gD2 and MOMP by Western blot analysis

Plasmids pEL-gD2 and pCOM2 were separately introduced into V. cholerae 01 strain H1 by electroporation, and clones containing the respective plasmids were isolated. The expression of gD2 or MOMP by the Vibrio clones was evaluated by Western blot analysis as previously described (Eko, 2000) using the mouse anti-gD2 monoclonal (7F12H5) (Austral Biologicals, CA) or anti-MOMP (MoPn 40, a generous gift from Dr Sukumar Pal) antibodies, respectively.

Production of rVCG coexpressing gD2 and MOMP

Production of rVCG was carried out essentially as described previously (Eko, 2003). Briefly, competent H1 cells (harbouring plasmid pEL-gD2, or pCOM2) were cotransformed with the lysis plasmid pDKLO1 (Kloos, 1994), and bacterial cells were grown at 37°C to an A600 nm of 0.30. The expression of recombinant proteins was induced by addition of isopropyl-β-d-thiogalactopyranoside (IPTG) to a final concentration of 2 mM, and cell lysis was achieved by the addition of 3-methyl benzoate to induce gene E expression. After lysis, cultures were harvested, washed with PBS or a low ionic buffer, and lyophilized. The efficiency of E-mediated killing of vibrios was estimated by plating serial dilutions of samples on brain heart infusion (BHI) agar as previously described (Eko, 1994). Results indicated a 100% killing efficiency (i.e. no CFU were observed on plates at all dilutions). Lyophilized VCGs were weighed, and the number of CFU mg−1 of VCG was estimated based on the total number of CFU in the culture medium at the highest absorbance attained prior to lysis. Ghost preparations were stored at room temperature until used.

Mice and Immunization protocol

Five- to 8-week-old female C57BL/6 mice (Jackson Laboratory) were used for all experiments. The animals were housed in laminar flow racks under pathogen-free conditions at a constant temperature of 24°C with a cycle of 12 h of light and 12 h of darkness and were fed mouse chowder and water ad libitum. Mice were otherwise treated in accordance with IACUC (Institutional Animal Care and Use Committee), AALAC (American Association for Laboratory Animal Care) and NIH (National Institutes of Health) guidelines. Immunizations were carried out using a 1-mL syringe fitted with a 27-guage needle. Groups of 10 animals were vaccinated intramuscularly (IM) with lyophilized rVCG or VCG alone as follows: groups 1 and 2 received 3 mg of rVCG-MOMP or rVCG-gD2 per animal, respectively. Group 3 received a mixture of rVCG-MOMP and rVCG-gD2 (rVCG-MOMP+gD2), made up of 1.5 mg of each component, while group 4 served as the negative control and received 3 mg of VCG alone. Each dose was given in 50 μL of PBS per animal. The vaccine dose was formulated such that 1 mg of lyophilized rVCG or VCG corresponded to about 2 × 109 CFU. All immunizations were administered under phenobarbitol anesthesia (Ramsey et al., 1988), and animals were boosted twice at two weekly intervals.

Measurement of antichlamydial mucosal and systemic IgA and IgG antibodies

Two weeks after the last immunization, animals were bled by periorbital puncture and the serum pooled for each group. Mucosal secretions (vaginal samples) were collected by washing the vagina of each mouse with 100 μL of PBS (pH 7.2) and pooled. Trypsin Inhibitor (10 μg mL−1, Sigma) and EDTA (5 × 10−4 M, Sigma) were added to the samples and centrifuged at 10 000 g for 10 min at 4°C to remove the debris. Supernatants were collected and 10−3 M phenylmethylsulfonyl fluoride (Sigma) and 0.01% sodium azide (Sigma) were added. Samples were stored at −80°C until analysed. The sera and vaginal washes were titrated for the detection of anti MOMP and gD2 antibodies (secretory IgA and total IgG2a) by an indirect enzyme-linked immunosorbent assay (ELISA) in which plates were coated with UV-inactivated chlamydial or HSV-2 antigen. Briefly, Maxisorb 96-well plates (Costar) were coated overnight with 10 μg mL−1 of chlamydial or HSV-2 antigen in 100 μL of PBS at 4°C. For generating a standard calibration curve, wells were similarly coated in triplicate with IgA or IgG2a standard (0.0, 12.5, 25, 50. 100, 250, 500 and 1000 ng mL−1). After washing (PBS−0.05% Tween 20), plates were blocked with 1% bovine serum albumin containing 5% goat serum in PBS and then incubated with 100 μL of serum or 50 μL of vaginal wash in twofold serial dilutions at 37°C for 2 h. Plates were again washed and incubated with 100 μL of horseradish peroxidase-conjugated goat antimouse IgA or IgG2a (Southern Biotechnology Associates, Inc., Birmingham, Al) for 1 h at room temperature. Peroxidase substrate, 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was added, and the absorbance associated with colour development was measured at 490 nm on a Spectra Max 250 Microplate Autoreader (Molecular Devices Corp., Sunnyvale, CA). Results generated simultaneously with the standard curve display data sets, corresponding to absorbance values, as mean concentrations (ng mL−1)±SDs and represent the mean of triplicate wells for each sample set.

Local mucosal and systemic antigen-specific T cell responses

Eight weeks after the last immunization, animals designated for immunogenicity studies were sacrificed, and immune T cell-enriched cells were prepared from the lymphoid tissues (iliac lymph nodes (ILN) and spleens) by nylon wool enrichment procedure as previously described (Igietseme, 1998). Purified lymphoid cells contained at least 95% CD3+cells, as determined by fluorescence-activated cell sorting (FACS) analysis. Cells were cultured at 37°C in 5% CO2. The level of antigen-specific Th1 or Th2 response was assayed by measuring the antigen-specific interferon-γ (IFN-γ) or interleukin-4 (IL-4) production by each cell population, respectively. Briefly, purified T cells were plated in a serial doubling dilution in duplicate 96-well tissue culture plates at 2 × 105 cells well−1 and cultured with wild-type antigen presenting cells (APCs) and either chlamydial or HSV-2 antigen (10 μg mL−1) for 5 days. Background cultures contained 24 wells without APCs or antigen. Supernatants were harvested and assayed for cytokines using the Bio-Plex cytokine assay kit in combination with the Bio-Plex manager software (Bio-Rad, Hercules, CA). The mean and SD of all replicate cultures were calculated.

Protection studies

Groups of mice (20 mice per group) were immunized as described above and randomly assigned to two subgroups for subsequent chlamydial or HSV-2 challenge. Ten weeks after the last immunization, mice were injected subcutaneously with DePo Provera (Upjohn, Kalamazoo, MI) at a dosage of 2 mg per mouse in 50 μL of distilled water to synchronize the estrus cycle. Five days after progesterone administration, animals were anesthetized intraperitoneally with 3 mg ketamine clorhydrate (Parke-Davis) and 0.3 mg xylacin (Bayer), and challenged intravaginally with 107 IFU of live C. trachomatis serovar D as previously described (Eko, 2003) or 4 × 104 PFU (100 LD50) of HSV-2 SR 333. The lethal dose 50% (LD50) in mice of HSV-2 SR 333 was determined by intravaginal infection in a preliminary experiment to be 4 × 102 PFU. After challenge, animals were monitored daily for signs of illness, indicated by ruffled fur, arched backs, feeble movements, vaginal inflammation, hind limb paralysis, and death, and the survival rates were calculated. Animals showing signs of severe inflammation and paralysis were promptly euthanized. Vaginal swabs were collected every 3 days for 27 days following challenge, and C. trachomatis or HSV-2 was isolated from swabs in tissue culture by standard methods (Ramsey et al., 1988; Slomka, 1998). The experiment was repeated twice.

Statistical analysis

Data from multiple experiments were expressed as mean±SDs. Statistical analyses were performed with the sigma stat statistics program (SPSS Inc., Chicago, IL). Data were analysed with Student's t-test at probability values of <0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Construction of vaccine vectors expressing chlamydial and HSV-2 recombinant proteins

Construction of plasmid pEL-gD2 was accomplished by placing the gD2 coding region without the signal peptide between and in frame with the E′ and L′ anchors, while pCOM2 was constructed by placing the Omp1 coding region, under the transcriptional control of the lac promoter and in frame with the LacZ′ and E′ anchors (Fig. 1). The full-length gD2 and ompl genes were thus expressed as E′-L′ and lacZ′-E′ fusion proteins, respectively. DNA sequencing confirmed that the cloned genes were in frame with the fusion anchors, and that the integrity of the plasmid constructs was maintained. Expression of the recombinant proteins (rgD2 and rMOMP) was confirmed by Western blot analysis using mouse mAbs to gD2 or MOMP. These results confirmed that transformants harbouring either pEL-gD2 or pCOM2 could efficiently express the HSV-2 or chlamydial proteins, respectively (data not shown).

image

Figure 1.  Construction of the expression plasmid vectors pEL-gD2 and pCOM2. The coding regions of gD2 and omp1, minus the leader sequences, were amplified by PCR from the chromosomal DNA of HSV-2 strain 333 and Chlamydia trachomatis serovar D, respectively. The gD2 PCR product was cloned into the membrane-targetting vector, pKSEL5-2, at the XbaI/HindIII sites to generate the plasmid pEL-gD2. The Omp1 expression vector, pCOM2, was constructed by inserting the entire Omp1 coding region, minus the 22-aa signal peptide, between and in frame with the E′ and L′ anchor sequences at the SalI and PstI restriction sites. Expression of gD2 and omp1 in both vectors is under the transcriptional regulation of the lac promoter (lacpo).

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Local mucosal and humoral antibody responses to rVCG vaccines

To assess the immunogenicity of the vaccines, IgA and IgG2a responses induced 2 weeks following immunization were measured by titrating serum and vaginal secretions of vaccinated mice against either chlamydial or HSV-2 antigen. There were no detectable antibody levels in the serum and vaginal washes of mice immunized with VCG alone (control). A baseline value of 0.05 ng mL−1 represented background levels based on antibody response in control mice. Significant levels of antigen-specific secretory IgA but marginal levels of IgG2a were detected in the vaginal secretions of mice immunized with the various vaccine constructs (Fig. 2). In contrast, significant levels of antigen-specific IgG2a were induced in serum, while only basal levels of systemic IgA were elicited (Fig. 3). The mucosal secretory IgA antibody levels were significantly higher (P<0.05) in mice vaccinated with the single vaccines than in those that received the multivalent combination vaccine. This may be directly related to the amount of antigen present in each formulation, as the combination vaccine contained 50% of the amount of each antigen present in the single vaccine constructs. However, there was no statistical difference between the levels of IgG2a in the serum of mice immunized with either single or combination vaccine (Fig. 3).

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Figure 2.  Induction of secretory antibodies in the genital mucosa 2 weeks after immunization with rVCG vaccines. Groups of female C57BL/6 mice were immunized intramuscularly and boosted twice, at two weekly intervals, with either rVCG-MOMP or rVCG-gD2 or a mixture of both or VCG alone. Vaginal washes were performed with 100 μL of PBS 2 weeks after immunization and stored at −80°C until assayed. The levels of (a) Chlamydia-specific or (b) HSV-2-specific secretory IgA and IgG2a antibodies in vaginal washes against the respective antigens were measured by a modified ELISA procedure. Results generated simultaneously with a standard curve display data sets, corresponding to absorbance values, as mean concentrations (ng mL−1)±SD and represent the mean of triplicate wells for each sample set. The difference between the levels of secretary IgA elicited by the single and combination vaccines was statistically significant (*) P<0.05.

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image

Figure 3.  Induction of systemic antibodies in sera 2 weeks after immunization with rVCG vaccines. Female C57BL/6 mice were vaccinated as described in Fig. 2. Sera were collected from the mice 2 weeks after immunization and stored at −80°C until assayed. The levels of (a) Chlamydia-specific or (b) HSV-2-specific secretory IgA and IgG2a antibodies in serum samples against the respective antigens were measured by a modified ELISA procedure. Results generated simultaneously with a standard curve display data sets, corresponding to absorbance values, as mean concentrations (ng mL−1)±SD and represent the mean of triplicate wells for each sample set. The difference between the levels of systemic IgG2a elicited by the single and combination vaccines was not statistically significant (P>0.05).

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Immunization with rVCG expressing HSV-2 and chlamydial proteins induced a Th1-type immune response

Analysis of immunogenicity at 8 weeks postimmunization revealed that both vaccine constructs as well as the multivalent formulation induced significantly elevated local mucosal antigen-specific Th1 responses, detectable in the ILN draining the genital tissues (P<0.05). Levels of IFN-γ produced by immune T cells, as a measure of Th1 response, are shown in Fig. 4. The level of mucosal Th1 immune response induced by the multivalent combination vaccine containing both gD2 and MOMP (rVCG-MOMP+gD2) was lower than that of the single subunit vaccines (rVCG-MOMP or rVCG-gD2). Significantly higher IFN-γ levels were also produced by splenic T cells from mice immunized with the rVCG subunit vaccines compared with those of mice immunized with VCG alone (P<0.001) (Fig. 5). The amounts of IFN-γ produced by both mucosal and systemic immune T cells in the response to restimulation by antigen from mice immunized with the rVCG-MOMP+gD2 combination vaccine were comparable (P>0.05) (Figs 4 and 5). Levels of Chlamydia- or HSV-2-specific IL-4 produced by ILN and splenic T cells from mice vaccinated with the various vaccines remained at baseline (data not shown).

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Figure 4.  Induction of local genital Chlamydia-specific and HSV-2-specific T cell responses by rVCG. Groups of mice were immunized as described in Fig. 2. The local mucosal Thl response was assessed, 8 weeks after immunization, by measuring the response of Chlamydia-specific and HSV-2-specific IFN-γ-secreting T cells from the genital tract tissue and iliac lymph nodes draining the genital tract of immunized mice. The amounts of IFN-γ contained in culture supernatants derived from cultured T cells stimulated with chlamydial antigen and controls were measured using a Bio-Plex cytokine assay kit in combination with the Bio-Plex manager software. The concentration of the cytokine in each sample was obtained by extrapolation from a standard calibration curve generated simultaneously. Data were calculated as the mean values (±SD) of triplicate cultures for each experiment. The control cultures without antigen did not contain detectable levels of IFN-γ and so the data were excluded from the results. The amount of IFN-γ induced by the bivalent (multidisease) combination vaccine was statistically different from that induced by the single subunit vaccines (P<0.05).

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Figure 5.  Induction of antigen-specific systemic T cell response by rVCG. Groups of mice were immunized as described in Fig. 2 and systemic IFN-γ response was assessed at 8 weeks postimmunization. The level of Thl response induced was determined by measuring the response of chlamydial-specific IFN-γ-secreting T cells from spleen cells of immunized mice. The amounts of IFN-γ contained in culture supernatants derived from cultured T cells stimulated with antigen and controls were measured using a Bio-Plex cytokine assay kit in combination with the Bio-Plex manager software. The concentration of the cytokine in each sample was obtained by extrapolation from a standard calibration curve generated simultaneously. Data were calculated as the mean values (±SD for triplicate cultures for each experiment. The control cultures without antigen did not contain detectable levels of IFN-γ and so the data were excluded from the results. The difference between the levels of systemic IFN-γ induced by the rVCG-MOMP and multidisease vaccines at week 8 postimmunization was not statistically significant (P>0.05).

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Efficacy of rVCG vaccines in mice against intravaginal challenge with viable C. trachomatis and HSV-2

Eleven weeks after the last immunization, the animals were challenged intravaginally with 107 IFU of live C. trachomatis serovar D. Mice immunized with the single chlamydial (rVCG-MOMP) and combination (rVCG-MOMP+rVCG-gD2) vaccines were highly resistant to infection, as indicated by the low number of chlamydial IFUs shed and the time taken to resolve the infection, compared with the group that received the single HSV-2 vaccine (rVCG-gD2) or VCG alone (control) (Fig. 6). While all the mice immunized with the rVCG-MOMP vaccine had completely resolved the infection within 2 weeks of challenge, some of the mice immunized with the combination vaccine remained infected at this time. In addition, none of the mice with rVCG-gD2 were protected, as evidenced by the high number of chlamydial IFUs shed up to 21 days postchallenge (Fig. 6).

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Figure 6.  Efficacy of protective immunity against Chlamydia challenge following immunization of mice with the rVCG vaccines. Groups of mice were vaccinated as described above and then challenged intravaginally, 11 weeks later, with 107 IFU of live C. trachomatis serovar D. Infections were monitored by cervicovaginal swabbing of individual animals every 3 days, and Chlamydia was isolated from swabs in tissue culture. The experiment was repeated twice with similar results. The data show the mean recoverable IFUs, expressed as log10 IFU mL−1, for each experimental group±SD. Student's t-test was used to compare differences between control and experimental groups at P<0.05.

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To ascertain the induction of protective antiviral immunity, immunized mice were challenged intravaginally with a high dose (100 LD50) of the homologous HSV-2 SR 333 11 weeks after the last immunization. All the animals immunized with rVCG-MOMP and VCG (control) became ill upon challenge, as indicated by their ruffled fur, arched backs, feeble movements, and swollen red vulva. They showed herpetic lesions and paralysis of hind limbs prior to death. About 60% of the mice in this group died 9 days after inoculation with the wild-type virus (Fig. 7), and those that became very ill at different times after challenge were promptly euthanized. Although three out of 10 (30%) mice immunized with the combination vaccine showed mild inflammation in the vaginal area, no lesions were observed in animals immunized with the single HSV-2 vaccine. Both the single HSV-2 and combination vaccines protected immunized mice against challenge with HSV-2, except for two mice immunized with the combination vaccine that died on day 21 postchallenge (Fig. 7). In contrast, animals immunized with either the single chlamydial vaccine or the control VCG were not protected and died within 18 days postchallenge.

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Figure 7.  Protective efficacy against HSV-2 challenge following immunization of mice with the rVCG vaccines. Groups of mice were vaccinated as described above and then challenged intravaginally with 4 × 104 PFU (100 LD50) of HSV-2 SR 333 11 weeks later. Animals were monitored daily and the survival rates were calculated. The experiment was repeated and gave similar results. The results are presented as the percentage of mice that survived over time. Student's t-test was used to compare differences between control and experimental groups at P<0.05.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The novel rVCG delivery platform, with its inherent adjuvant properties, offers an attractive approach for the induction of cellular and humoral immune responses directed against target antigens derived from single (Eko, 2003, 2004) or multiple pathogens. The pattern and magnitude of specific immune responses elicited after immunization often serve as indicators of protective immunity. However, the fundamental test of a vaccine is the ability to provide protection against challenge with a live pathogen following immunization. The VCG delivery vector together with the mouse genital infection model provided a convenient approach for evaluating the immunogenicity and protective efficacy of the bivalent combination vaccine formulation comprising rVCG expressing chlamydial MOMP and HSV-2 glycoprotein D. The results obtained are encouraging, because mice immunized with the combination vaccine were prophylactically protected from genital challenge with high doses of live Chlamydia and HSV-2.

An effective combination vaccine against Chlamydia and HSV-2 will most probably need to elicit effective Th1 cell-mediated immune responses as well as accessory or neutralizing antibodies (Rank, 1994; Cotter, 1997; Perry et al., 1997; Su, 1997). Significant levels of Th1 responses were induced following immunization, as indicated by the production of high levels of IFN-γ compared with IL-4 levels that remained unchanged up to 8 weeks postimmunization. IFN-γ has been shown to be involved in the clearance of HSV-2 from the vaginal mucosa of nonimmune mice and in resistance to reinfection in immune mice (Smith, 1994; Milligan & Bernstein, 1997; Milligan et al., 1998). The magnitude of IFN-γ secreted by mucosal immune T cells appeared to depend on the dose of the antigen. This is indicated by a decrease in the amount of IFN-γ secreted by T cells from mice immunized with the combination vaccine; this formulation contains 50% of the antigen dose present in the single subunit vaccines. High IFN-γ and low IL-4 levels are indicative of a Th1 response (Maggi, 1992; Bradley et al., 1995; Igietseme et al., 2003), which has previously been correlated with a degree of protection against both Chlamydia and HSV infections (Rank, 1994; Cotter, 1997; Perry et al., 1997; Su, 1997) (Koelle, 1998; Sin, 1999).

Significant amounts of the Th1-associated antibodies, IgA and IgG2a were also detected in the genital tracts and serum of mice immunized with the combination vaccine compared with the VCG-immunized controls. The role of humoral immunity in the control of genital chlamydial and herpes infections is yet to be completely delineated. Recent studies suggest that the predominant role of antibodies in chlamydial clearance is in resistance to reinfection, by enhancing the uptake, processing and presentation of chlamydial antigens by APCs for rapid Th1 activation and clearance of infection (Moore, 2002) – a finding that may also be applicable to other intracellular pathogens, including HSV-2. Antibodies may mediate protection by blocking the initial attachment of the pathogen to epithelial cells, thereby limiting dissemination to distant sites enhancing chlamydial clearance (Igietseme, 2004). Previous studies indicated that the level of protective immunity conferred by rVCG-based vaccines is a function of the ability of the vaccine to induce a high frequency of local mucosal Th1 cells after primary immunization (Eko, 2003, 2004). Thus, a vaccine delivery strategy that effects the induction of a fast and vigorous Th1 response following an infection will rapidly arrest the replication of the infecting organism, clear the infection, and prevent the establishment of a latent infection. If, on the other hand, the Th1 response is inadequate or suboptimal, there will be delayed clearance of the pathogen that may lead to the establishment of a latent or persistent infection.

The presence of Chlamydia- and HSV-2-specific immune effectors in the vaginal secretions of rVCG-vaccinated mice would indicate that IM immunization with these vaccines induces effective humoral- and cell-mediated immune responses that are targetted to the genital tract, in addition to other sites. Our results also show that immunization with the combination vaccine formulation was sufficient to inhibit chlamydial shedding in the vaginal tract or to prolong the survival of immunized mice following HSV-2 challenge. However, immunization with the combination vaccine led to a moderate level of protection against virulent Chlamydia and HSV-2 infections, suggesting that the protection afforded by the multivalent combination vaccine may also be dose-dependent. Our results using gD2 delivered by rVCG are in agreement with the results of other authors, who have reported the induction of protective immune responses and survival after challenge when mice were immunized with a plasmid encoding the full-length HSV-2 gD2 protein (Cooper, 2004; Natuk, 2006).

This study highlights three principal findings: (i) that rVCG could serve as efficient vehicles for the delivery of a combination vaccine targetted against multiple diseases, (ii) that a rVCG-delivered multivalent combination vaccine formulation could induce high levels of protective immune responses against multiple pathogens with no apparent impairment of the immune response to the individual vaccine components, and (iii) that rVCG are effective adjuvants inducing both T cell and humoral immune responses. The degree of protection provided by the rVCG vaccines in the acute phase of the challenge was quite remarkable when compared with other immunizations with subunit antigens without added adjuvants. The data demonstrate the possibility of developing a multivalent combination vaccine that can simultaneously protect against Chlamydia and HSV-2 infections. The study highlights the use of the VCG platform as a novel approach for designing combination vaccines against multiple pathogens.

In conclusion, we have shown that a rVCG-based combination vaccine comprising chlamydial MOMP and gD2 of HSV-2 elicited adequate Th1-associated immune effectors following immunization of mice. In addition, immunized mice were simultaneously protected from genital challenge with high doses of live Chlamydia and HSV-2. It will be observed that although the combination vaccine was formulated to contain 50% of the dose of the individual vaccines, it could still induce significant protection against challenge with the individual pathogens. The lower antigen dose in the combination vaccine was more obvious in the induction of local genital mucosal secretary IgA antibodies compared with systemic IgG2a antibodies. While the results obtained with this combination vaccine formulation are quite promising, future studies will ensure the use of comparable doses between the single and combination vaccine formulations.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Dr Sukumar Pal for kindly providing the monoclonal antibodies MoPn 40 used for Western blot analysis and Dr Jorge Benitez for useful suggestions. This work was funded by a Public Health Service grant GM 08248 from the National Institutes of Health. The investigation was conducted in a facility constructed with support from Research Facilities Improvement Grant no. 1 C06 RR18386 from the National Center for Research Resources, National Institutes of Health.

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  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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