Plasmid DNA-expressed secreted and nonsecreted forms of herpes simplex virus glycoprotein D2 induce different types of immune responses.

Herpes simplex viruses (HSVs) are significant pathogens and major targets of vaccine development. Several attempts have been made to develop prophylactic and therapeutic vaccines for HSV types 1 and 2. Although these vaccines elicit strong humoral responses, the overall impact on pathology has been disappointing. An effective vaccine for HSV must induce both humoral and cellular immune responses. DNA vaccines are ideal candidates for HSV vaccines because they induce both types of immune responses. This study showed that the type of immune response generated by immunization with DNA vaccines is modulated by expression of various forms of an antigen, each with a different cellular localization. Expression of cell-associated forms of HSV-2 glycoprotein D (gD) induces primarily a Th1 response, whereas expression of secreted gD results in a Th2 response. Immunization with plasmids expressing different forms of the antigen may increase the efficacy of a vaccine.

Induction of HSV-gD2 specific CD4(+) cells in Peyer's patches and mucosal antibody responses in mice following DNA immunization by both parenteral and mucosal administration.

A DNA vaccine encoding glycoprotein D (gD) of herpes simplex virus type 2 (pHSV-gD2) was injected via parenteral and mucosal routes to determine the optimal route of delivery for immune stimulation. Generation of distal mucosal immunity following parenteral vaccination was also evaluated. While all routes of DNA vaccine administration resulted in systemic cellular and humoral responses, the intra-muscular (i.m.) and intra-dermal (i.d.) routes of delivery produced the highest responses. Furthermore, i.m. and i.d. routes produced mucosal humoral responses that were comparable to those obtained via mucosal routes. Specific pHSV-gD2 PCR signals were detected in the Peyer's patches (PP) within hours following vaccination and antigen specific IgA was detected in secretions and supernatants from gut fragment cultures. Furthermore, antigen specific CD4(+) cells were found in PP. Collectively these results suggest that the DNA vaccine stimulated a response in the PP, a major inductive site for mucosal responses.

IL-12 gene as a DNA vaccine adjuvant in a herpes mouse model: IL-12 enhances Th1-type CD4+ T cell-mediated protective immunity against herpes simplex virus-2 challenge.

IL-12 has been shown to enhance cellular immunity in vitro and in vivo. Recent reports have suggested that combining DNA vaccine approach with immune stimulatory molecules delivered as genes may significantly enhance Ag-specific immune responses in vivo. In particular, IL-12 molecules could constitute an important addition to a herpes vaccine by amplifying specific immune responses. Here we investigate the utility of IL-12 cDNA as an adjuvant for a herpes simplex virus-2 (HSV-2) DNA vaccine in a mouse challenge model. Direct i.m. injection of IL-12 cDNA induced activation of resting immune cells in vivo. Furthermore, coinjection with IL-12 cDNA and gD DNA vaccine inhibited both systemic gD-specific Ab and local Ab levels compared with gD plasmid vaccination alone. In contrast, Th cell proliferative responses and secretion of cytokines (IL-2 and IFN-gamma) and chemokines (RANTES and macrophage inflammatory protein-1alpha) were significantly increased by IL-12 coinjection. However, the production of cytokines (IL-4 and IL-10) and chemokine (MCP-1) was inhibited by IL-12 coinjection. IL-12 coinjection with a gD DNA vaccine showed significantly better protection from lethal HSV-2 challenge compared with gD DNA vaccination alone in both inbred and outbred mice. This enhanced protection appears to be mediated by CD4+ T cells, as determined by in vivo CD4+ T cell deletion. Thus, IL-12 cDNA as a DNA vaccine adjuvant drives Ag-specific Th1 type CD4+ T cell responses that result in reduced HSV-2-derived morbidity as well as mortality.

Development and maintenance of the gut-associated lymphoid tissue (GALT): the roles of enteric bacteria and viruses.

GALT can be subdivided into several compartments: (a) Peyer's patches (PP); (b) lamina propria (LP); and (c) intraepithelial leukocyte (IEL) spaces. The B-cell follicles of PP are quiescent in neonatal and germ-free (GF) adult mice. Germinal centers (GC), including sIgA+ blasts, appear in the B follicles of formerly GF adult mice about 10-14 days after monoassociation with various gut commensal bacteria. The GC wax and wane over about a 3-week period, although the bacterial colonizers remain in the gut at high density. Neonatal mice, born of conventionally reared (CV), immunocompetent mothers, display GC reactions in PP postweaning, although pups of SCID mothers display precocious GC reactions at about 14 days of life. Normally, gut colonization of neonates with segmented filamentous bacteria (SFB) leads to explosive development of IgA plasmablasts in LP shortly after weaning. Commensal gut bacteria and the immunocompetency of mothers also appears to control the rate of accumulation of primary B cells from "virgin" B cells in neonates. Enteric reovirus infection by the oral route can cause the activation of CD8+ T cells in the interfollicular regions of PP and the appearance of virus-specific precursor cytotoxic T lymphocytes (pCTL) in the IEL spaces. Such oral stimulation can also lead to "activation" of both CTL and natural killer (NK) cells in the IEL spaces. More normally, colonization of the gut with SFB also leads to similar activations of NK cells and "constitutively" cytotoxic T cells.

Commensal enteric bacteria engender a self-limiting humoral mucosal immune response while permanently colonizing the gut.

We have employed a germfree mouse model to study the development and persistence of a humoral mucosal immune response to a gram-negative murine commensal organism, Morganella morganii. M. morganii bacteria rapidly colonize the gut, resulting in hypertrophy of Peyer's patches (PP), including germinal center reactions (GCR), and the development of specific immunoglobulin A (IgA) responses detected in vitro in PP fragment cultures and by ELISPOT assays of lamina propria cells. The GCR peaks 14 days after infection and begins to wane thereafter. Upon colonization, the organisms successfully translocate to the mesenteric lymph node and spleen, but the number of translocating bacteria begins to drop with the onset of a specific IgA response. A clonal B-cell microculture technique was used to determine the frequency of specific IgA plasmablasts and IgA memory cells. The frequencies of preplasmablasts were seen to be higher in the earlier stages of germinal center development, whereas the frequencies of antigen-specific memory cells appeared to remain at a relatively constant level even after 193 days postmonoassociation. We suggest that a successful secretory IgA response can attenuate chronic stimulation of GCR even though the bacteria persist in the gut. The observed developing hyporesponsiveness to a chronically present commensal organism may be relevant to the use of bacterial vectors for mucosal immunization.

Ligation of CD45 on B cells can facilitate production of secondary Ig isotypes.

The possibility that isotype switching in B cells may be affected by engagement of the CD45 molecule on B cells has been investigated in microcultures containing limiting numbers of B cells and nonlimiting numbers of both alloreactive Th cells and purified dendritic cells (DC). Addition of Abs to the B cell-specific isoform, B220, to the microcultures leads to an increase in the proportion of B cell clones that secrete secondary Ig isotypes. In the presence of anti-CD45 Ab, microculture wells show a 39% frequency of secondary isotypes (560/1440) compared with a 11% frequency in control microcultures (89/780). Cross-linking appears to enhance this effect. Even in cultures of B cells and DC without T cells, addition of anti-B220 induces isotype switching in a significant number of microwells. Cross-linking and capping B220 molecules results in co-capping of surface Ig and MHC class II molecules. The results suggest that signal transduction through the CD45 molecule may affect pathways involved in isotype switching.

Major histocompatibility complex restriction of T-cell suppression of immune response to mycobacteria.

In earlier work, intraperitoneal (i.p.) immunization with Mycobacterium vaccae was shown to generate a T-suppressor (Ts) response but intradermal (i.d.) immunization did not. We have now studied the major histocompatibility complex (MHC) restriction of this Ts response. The ability of C57BL/6 (H-2b), BALB/c (H-2d), and the (C57BL/6 x BALB/c) F1 mice to generate suppression after i.p. immunization with 10(8) killed M. vaccae was investigated. The BALB/c and the F1 mice generated suppression, but the C57BL/6 mice failed to do so. The suppression could be ascribed to Lyt-2+, L3T4- antigen-specific T cells. The F1 suppressors generated after i.p. immunization could suppress the generation of T-cell responses to i.d. immunization with M. vaccae in the parental BALB/c but not in the C57BL/6 mice. Monoclonal anti-I-A antibody could suppress the antigen-induced proliferative response of mice primed i.d. with M. vaccae. In contrast, monoclonal anti-I-E antibody enhanced antigen-specific proliferation of spleen cells primed i.p. with M. vaccae. The suppressors generated by i.p. priming of mice with M. vaccae could also suppress the in vitro antigen-induced proliferative response of i.d.-primed spleen cells; the suppression could be blocked by anti-I-E antibody. Thus, the T-cell-mediated suppression in the above experimental model was I-E restricted. The inability of the C57BL/6 mice to generate suppression after i.p. immunization with M. vaccae was ascribed to the lack of I-E expression by mice of H-2b strain.

Pathogenesis of route-related variation in T-suppressor response on immunization with mycobacteria.

The route of immunization was observed to play a significant role in deciding the outcome of immunization with killed mycobacterial vaccines. Earlier we reported that the slow growers were immunogenic by both the intraperitoneal (i.p.) and intradermal (i.d.) routes. In contrast, the rapid growers were immunogenic by the i.d. route only. Both rapid and slow growers generated the classical, antigen-specific Lyt-2 positive, T-cell-mediated suppression after i.p. immunization but not after i.d. immunization. Thus, in the case of the slow growers, T-cell-mediated suppression was only a component of the immune response generated after i.p. immunization. In contrast, in the case of Mycobacterium vaccae and the other rapid growers, the T-cell-mediated suppression was the predominant response with i.p. immunization. The T-cell-mediated suppression generated by i.p. immunization exhibited crossreactivity, the spectrum of which was dependent upon the dose of the immunization.

Route-related variation in immunogenicity of mycobacteria.

The route of immunization was observed to play a significant role in deciding the T-cell response to immunization with killed mycobacterial vaccines. Slow-growing mycobacteria were found to be immunogenic by both the intraperitoneal (i.p.) and intradermal (i.d.) routes; rapid-growing mycobacteria were immunogenic by the i.d. route only. The nonresponder state following i.p. immunization with Mycobacterium vaccae could be corrected by treatment of the mice with poly I:poly C or indomethacin prior to immunization. Both poly I:poly C, an interferon inducer, and indomethacin, a prostaglandin inhibitor, are known to enhance the expression of major histocompatibility complex glycoproteins. Since they are so important in antigen preparation, it was concluded that the inability of mice to respond to M. vaccae by the i.p. route is likely due to defective presentation of the bacterial antigens by the antigen-presenting cells at the site, namely, the peritoneal macrophages. These findings are significant because M. leprae has been reported to be antigenically similar to M. vaccae, and the response of mice to i.p. immunization with both of these mycobacteria is very similar.

Variation in immunogenicity of mycobacteria: role of antigen-presenting cells.

The antigen-presenting efficiency of peritoneal cells and irradiated spleen cells was compared using Mycobacterium tuberculosis- and M. vaccae-primed T cells and corresponding sonicates as antigens in an in vitro lymphocyte transformation test. The presentation efficiency of irradiated spleen cells was reasonably good for both antigens. However, with peritoneal cells as the antigen-presenting cells, the proliferative response against only M. tuberculosis sonicate was good. Proliferation of M. vaccae-primed T cells was very poor when the antigen was presented by peritoneal cells. Poly I:poly C treatment of mice prior to harvesting the peritoneal cells resulted in distinct improvement in their efficiency to present M. vaccae sonicate; maximal proliferative response was obtained with peritoneal cells from mice receiving two and three doses of poly I:poly C 24 hr apart. Even paraformaldehyde-fixed peritoneal cells from poly I:poly C-treated mice gave an efficient M. vaccae-specific stimulation to primed T cells. Based on these data, it was concluded that failure of mice to respond to M. vaccae by intraperitoneal immunization is the result of the poor efficiency of presentation of M. vaccae antigen.

Role of antigen-presenting cells in variation in immunogenicity of mycobacteria.

The route of immunization was observed to play a significant role in deciding the outcome of immunization with killed mycobacterial vaccines. Whereas the slow growers were immunogenic by both intraperitoneal and intradermal routes, the rapid growers were immunogenic only by intradermal route. The non-responder state of mice to Mycobacterium vaccae by i.p. route of immunization could be corrected by prior treatment with poly I:poly C, an interferon inducer, or indomethacin, a prostaglandin inhibitor. Antigen-presenting efficiency of peritoneal and spleen cells were compared employing M. vaccae and M. tuberculosis H37Rv primed T cells and corresponding sonicates as antigens in an in vitro lymphocyte transformation test. Irradiated spleen cells presented both the antigens efficiently. However, with peritoneal cells as antigen-presenting cells, proliferative response against only M. tuberculosis was observed; proliferation of M. vaccae primed T cells was very poor. Peritoneal cells of poly I:poly C treated mice showed distinct improvement in their efficiency of presentation; even paraformaldehyde-fixed peritoneal cells gave an efficient stimulation with M. vaccae. The percentage of Ia-positive fraction in peritoneal cells was very low (5.95%) in comparison with spleen cells (38.37%). Poly I:poly C treatment resulted in increase in the Ia-positive cell fraction of the peritoneal cells to 24.5%.

Route-related variation in the immunogenicity of killed Salmonella enteritidis vaccine: role of antigen presenting cells.

In order to assess the role of the route of immunization on the immunogenicity of killed Salmonella vaccine, mice were immunized with killed S. enteritidis by intraperitoneal (i.p.) and intradermal (i.d.) routes. Whereas the former was non-immunogenic, the i.d. immunization generated an excellent delayed-type hypersensitivity response; further, i.p. immunization could even suppress the subsequent i.d. immunization. Since the peritoneal macrophages (MO) are known to be particularly low in Ia or MHC-class II antigens, so essential for antigen presentation, the non-immunogenicity by i.p. route was thought to be due to their poor presentation efficiency. Poly I: poly C, an interferon inducer, is known to enhance the MHC-class II expression; hence effect of poly I: poly C treatment on the immunogenicity of the killed vaccine by i.p. route was tested and indeed the non-immunogenicity was corrected. Poor efficiency of presentation of S. enteritidis antigen by peritoneal cells and its improvement by prior poly I: poly C treatment was further confirmed by in vitro lymphocyte transformation test using primed T cells and peritoneal cells from normal and poly I: poly C treated mice. Poly I: poly C treatment also enhanced expression of Ia antigens on peritoneal cells.