Mucosal immunogenicity of genetically detoxified derivatives of heat labile toxin from Escherichia coli

Using a fixed dose of antigen, the immune response to detoxified mutants of LT-WT following intranasal (i.n.), subcutaneous (s.c.) and oral (i.g.) immunisation has been studied. When given i.n., both LT-WT and mutant toxin, K63, generated significant levels of toxin-specific IgG in the serum, and the levels of IgA in nasal and lung lavages were greater than those induced by rLT-B. In comparison, i.g. immunisation of mice with a similar quantity of either LT-WT or K63 toxin induced barely detectable levels of IgG in the sera. However, if the amount of protein used for i.g. immunisation was increased tenfold, relatively good levels of toxin-specific IgG were induced in the sera by both LT-WT or K63. Low levels of toxin-specific IgA were also observed in intestinal washes from these mice. Western blotting of the sera, using the native toxin as an antigen, demonstrated the presence of both anti-A and anti-B subunit antibodies. Most significantly, toxin-neutralising antibodies were induced in the serum, with the strongest activity being induced by the LT-WT, an intermediate activity induced by mutant K63 and a lower response by rLT-B. Together, these data show that ADP-ribosyltransferase is not necessary for mucosal immunogenicity of these proteins, and that the i.n. route of immunisation is more effective than the i.g. route of immunisation for the generation of both systemic (IgG) and mucosal (IgA) immune responses.     

Use of intranasal IL-12 to target predominantly Th1 responses to nasal and Th2 responses to oral vaccines given with cholera toxin

We have investigated the effects of IL-12 and cholera toxin (CT) on the immune response to tetanus toxoid (TT) given by intranasal or oral routes. CT inhibited IL-12-induced IFN-gamma secretion both in vivo and in vitro. Intranasal administration of IL-12 to mice nasally immunized with the combined vaccine of TT and CT resulted in increased TT-specific IgG2a and IgG3 Abs, while IgG1 and IgE Ab responses were markedly reduced. This shift of the CT-induced immune response toward Th1 type was associated with TT-specific CD4+ T cells secreting IFN-gamma and reduced levels of Th2-type cytokines (i.e., IL-4, IL-5, IL-6, and IL-10). In contrast, intranasal IL-12 enhanced the CT-induced serum IgG1 and IgE Ab responses in mice given the combined vaccine orally. IFN-gamma secretion by TT-specific CD4+ T cells was also enhanced; however, Th2-type cytokine responses were predominant. Mucosal secretory IgA responses to oral or nasal vaccines were not affected by intranasal IL-12. Thus, intranasal IL-12 delivery influences Th cell subset development in mucosal inductive sites that are dependent on the route of vaccine delivery.     

Protection against anthrax toxin by vaccination with a DNA plasmid encoding anthrax protective antigen

A DNA vaccine encoding the immunogenic and biologically active portion of anthrax protective antigen (PA) was constructed. Spleen cells from BALB/c mice immunized intramuscularly with this vaccine were stimulated to secrete IFN gamma and IL-4 when exposed to PA in vitro. Immunized mice also mounted a humoral immune response dominated by IgG1 anti-PA antibody production, the subclass previously shown to confer protection against anthrax toxin. A 1:100 dilution of serum from these animals protected cells in vitro against cytotoxic concentrations of PA. Moreover, 7/8 mice immunized three times with the PA DNA vaccine were protected against lethal challenge with a combination of anthrax protective antigen plus lethal factor.     

Humoral and cellular immune responses in the murine respiratory tract following oral immunization with cholera toxin or Escherichia coli heat-labile enterotoxin

Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are the strongest mucosal immunogens identified to date and are also good adjuvants when given orally together in combination with unrelated antigens. We used these potent immunogens to monitor local and systemic immune responses following oral immunization of BALB/c mice, and compared their action on the following: (a) immunoglobulin production rates (IgG, IgM and IgA) in mucosal inductive (Peyer's patches-PPs), effector (intestinal lamina propria-LP, respiratory tract) and systemic (spleen) sites; (b) analysis of systemic antigen-specific antibodies (IgG subclasses, IgA and IgE); (c) time monitoring of fecal anti-CT and anti-LT antibodies, and (d) in vivo relevance of interleukin-6 (IL-6) to mucosal responses. Both mucosal immunogens elicited specific antibody responses (IgA, IgG) not only in the gastrointestinal tract (PP's and intestinal LP), but also in the respiratory tract and spleens of orally immunized mice. These mucosal responses were accompained by elevated secretion of IL-6 in all investigated tissues, indicating involvement of this cytokine in B-cell maturation processes. Furthermore, oral immunization with CT and LT induced elevated serum titers of IgG1 followed by IgG2a, IgG2b, IgG3 and IgA, while high antigen-specific IgA and IgG1 responses were found in fecal extracts. These findings illustrate the action of orally administered CT and LT, respectively, on several humoral and cellular immune responses not only at the gastrointestinal tract, the application site, but also in distant mucosal effector sites such as the respiratory tract. These data suggest the potential use of these mucosal adjuvants in oral immunization strategies to improve the local immune response in remote mucosal tissues, in accordance with the concept of a common mucosa-associated immune system.     

Relationship of blood transfusion, post-operative infections and immunoreactivity in patients undergoing surgery for gastrointestinal cancer

The development of IgG subclass-specific antibody responses to Plasmodium berghei in spleen-chimeric rats were monitored to determine if there was any relationship between IgG subset profiles and resistance. Strongly immune eusplenic rats respond to challenge with P. berghei by producing high levels of parasite-specific IgG2a, IgG2b and IgG2c but only modest levels of IgG1. Splenectomy profoundly affects the antibody response to infection. Thus, in splenectomized immunized rats, which harbour a chronic parasitaemia of 1%, the IgG2a, IgG2b and IgG2c responses peak 1 week later than in eusplenic immunized rats although the size of the peak is similar. More marked effects are apparent in the IgG1 response, the magnitude of which is far greater in splenectomized immunized rats than eusplenic immunized rats. Similar antibody profiles are seen in splenectomized immunized rats transplanted with a naive spleen. In contrast, splenectomized naive rats receiving either a transplant of a spleen from an immune rat or a transfer of immune spleen cells have high levels of IgG2a, IgG2b and IgG2c but modest levels of IgG1. However, only the former group of rats completely clears the parasite, the latter maintaining a chronic 1% parasitaemia. Thus, although complete resistance to P. berghei is always associated with high levels of parasite-specific IgG2a, IgG2b and IgG2c plus modest levels of IgG1, this is not a sufficient set of conditions to guarantee complete immunity. The IgG subset profile may be related to cytokine production; IFN-gamma was detected in the sera of rats receiving spleens from rats immune to P. berghei (modest IgG1 responses) but not in rats receiving spleens from naive animals (pronounced IgG1 responses).     

Antibody-secreting cells specific for simian immunodeficiency virus antigens in lymphoid and mucosal tissues of immunized macaques

OBJECTIVES: To examine whether the route of immunization affects the induction of antibody-secreting cells (ASC) in the circulation of macaques. The distribution of ASC in the rectal mucosa and lymphoid tissues following challenge with simian immunodeficiency virus (SIV) was investigated. DESIGN: Macaques were immunized with recombinant SIV gp120 and p27 antigens by the targeted iliac lymph node (TILN) route of immunization or the nasal and rectal route, augmented by intramuscular immunization [naso-rectal intramuscular (NRI)]. The macaques were challenged with live SIV by the rectal route and ASC were assayed in the circulation before and after SIV challenge, and in the tissues removed at post-mortem. METHODS: ASC were examined in the circulation by Elispot assay. Mononuclear cells were prepared from peripheral blood, iliac and axillary lymph nodes and spleen. Rectal tissue was treated by enzyme digestion to elute mononuclear cells. RESULTS: TILN and NRI immunization induced circulating IgA and IgG ASC to both gp120 and p27. Following rectal challenge with SIV, TILN macaques were protected from infection whereas NRI route-immunized and unimmunized controls became infected. IgA ASC to p27 were increased significantly in the iliac lymph nodes of the TILN immunized macaques compared with unimmunized controls (P < 0.05). Only IgA ASC were found in the rectal mucosa of the immunized protected macaques but both IgA and IgG ASC were detected in the unimmunized infected macaques. Overall the number of IgG ASC specific for p27 was significantly higher in the infected NRI and control macaques than in the protected macaques (P < 0.02). A progressive increase in IgG but not IgA ASC was detected in the peripheral blood mononuclear cells of the unimmunized infected macaques. CONCLUSIONS: The results suggest that cells secreting IgA antibodies to p27 in the iliac lymph nodes of the TILN immunized macaques correlate significantly with protection from infection. The unimmunized infected macaques showed a progressive increase in IgG ASC in the peripheral blood after SIV challenge; this was found in the iliac and axillary lymph nodes and also in the spleen, suggesting that it is an immune response to the SIV infection.     

Intranasal immunization with CTL epitope peptides from HIV-1 or ovalbumin and the mucosal adjuvant cholera toxin induces peptide-specific CTLs and protection against tumor development in vivo

To evaluate the ability of mucosal immunization protocols using peptide immunogens to induce CTL responses, BALB/c and C57BL/6 mice were immunized intranasally (i.n.) with peptides corresponding to a known CTL epitope in HIV-1 glycoprotein 120 or OVA, respectively, and the mucosal adjuvant cholera toxin (CT). Intranasal immunization of BALB/c mice with a 10- or 15-amino acid peptide corresponding to a CTL determinant in HIV-1 glycoprotein 120 and CT induced peptide-specific CTLs in spleen cells that persisted through 35 days after the last immunization. Intranasal immunization of C57BL/6 mice with the octameric OVA peptide and CT produced similar results with detectable peptide-specific CTL in both the cervical lymph node and spleen. To test whether CTL induced by i.n. immunization with OVA peptide and CT were functional in vivo, groups of C57BL/6 mice were injected with E.G7-OVA tumor cells that express the OVA protein and monitored for tumor growth. Animals immunized i.n. with OVA and CT were protected against tumor development as efficiently as animals immunized by the potent CTL induction protocol of i.v. injection with OVA-pulsed dendritic cells. Intranasal immunization with peptides corresponding to known CTL epitopes and CT provides a noninvasive route of immunization for the induction of CTL responses in vivo.     

Comparative efficacy of experimental anthrax vaccine candidates against inhalation anthrax in rhesus macaques

The authors examined the efficacy of Bacillus anthracis protective antigen (PA) combined with adjuvants as vaccines against an aerosol challenge of virulent anthrax spores in rhesus macaques. Adjuvants tested included i) aluminum hydroxide (Alhydrogel), ii) saponin QS-21 and iii) monophosphoryl lipid A (MPL) in squalene/lecithin/Tween 80 emulsion (SLT). Animals were immunized once with either 50 micrograms of recombinant PA plus adjuvant, or with Anthrax Vaccine Adsorbed (AVA), the licensed human anthrax vaccine. The serological response to PA was measured by enzyme linked immunosorbent assay. Lymphocyte proliferation and serum neutralization of in vitro lethal toxin cytotoxicity were also assayed. In all vaccine groups, anti-PA IgM and IgG titers peaked at 2 weeks and 4-5 weeks postimmunization, respectively. Five weeks postimmunization, animals in all vaccine groups demonstrated PA-specific lymphocyte proliferation and sera that neutralized in vitro cytotoxicity. Six weeks after immunization, the animals were challenged by aerosol with approximately 93 LD50 of virulent anthrax spores. Animals were bled daily for 1 week to monitor bacteremia, and deaths were recorded. Anti-PA ELISA titers in all groups of immunized animals were substantially increased 2 weeks after challenge. One dose of each vaccine provided significant protection (> 90%) against inhalation anthrax in the rhesus macaques.     

Isotype profiles of Leishmania donovani-infected BALB/c mice: preferential stimulation of IgG2a/b by liposome-associated promastigote antigens

Leishmaniasis presents a complex spectrum of diseases and immunological manifestations depending upon both the species of the microorganism and the host it infects. BALB/c mice, which are homozygous for Lsh(s) on chromosome 1, are genetically susceptible to the visceralizing species of Leishmania. Infection of these mice with an Indian strain of Leishmania donovani showed a steady rise in the level of parasite burden in both the liver and the spleen to 24 wk. To investigate the immune responses determining the course of infection, we studied the relative levels of specific IgG, IgM, and IgA antibodies, and IgG isotypes, in the sera of diseased and protectively immunized mice at different periods of infection. IgG1 and IgG2a were stimulated in the control, infected, and immunized mice after parasite challenge. However, an early induction of IgG1 in the normal infected mice and stimulation of IgG2a and IgG2b isotypes prior to parasite challenge in liposome-antigen-immunized mice suggest that the elicitation of a particular subset of CD4+ T cells at the onset of disease may be responsible for either progression or resolution of infection.     

Mutants in the ADP-ribosyltransferase cleft of cholera toxin lack diarrheagenicity but retain adjuvanticity

Cholera toxin (CT), the most commonly used mucosal adjuvant in experimental animals, is unsuitable for humans because of potent diarrhea-inducing properties. We have constructed two CT-A subunit mutants, e.g., serine-->phenylalanine at position 61 (S61F), and glutamic acid-->lysine at 112 (E112K) by site-directed mutagenesis. Neither mutant CT (mCT), in contrast to native CT (nCT), induced adenosine diphosphate-ribosylation, cyclic adenosine monophosphate formation, or fluid accumulation in ligated mouse ileal loops. Both mCTs retained adjuvant properties, since mice given ovalbumin (OVA) subcutaneously with mCTs or nCT, but not OVA alone developed high-titered serum anti-OVA immunoglobulin G (IgG) antibodies (Abs) which were largely of IgG1 and IgG2b subclasses. Although nCT induced brisk IgE Ab responses, both mCTs elicited lower anti-OVA IgE Abs. OVA-specific CD4+ T cells were induced by nCT and by mCTs, and quantitative analysis of secreted cytokines and mRNA revealed a T helper cell 2 (Th2)-type response. These results now show that the toxic properties of CT can be separated from adjuvanticity, and the mCTs induce Ab responses via a Th2 cell pathway.     

Antifungal imidazoles block assembly of inducible NO synthase into an active dimer

The humoral immune responses to the D2 peptide of fibronectin-binding protein B (FnBP) of Staphylococcus aureus, expressed on the plant virus cowpea mosaic virus (CPMV), were evaluated after mucosal delivery to mice. Intranasal immunization of these chimeric virus particles (CVPs), either alone or in the presence of ISCOM matrix, primed CPMV-specific T cells and generated high titers of CPMV- and FnBP-specific immunoglobulin G (IgG) in sera. Furthermore, CPMV- and FnBP-specific IgA and IgG could also be detected in the bronchial, intestinal, and vaginal lavage fluids, highlighting the ability of CVPs to generate antibody at distant mucosal sites. IgG2a and IgG2b were the dominant IgG subclasses in sera to both CPMV and FnBP, demonstrating a bias in the response toward the T helper 1 type. The sera completely inhibited the binding of human fibronectin to the S. aureus FnBP. Oral immunization of the CVPs also generated CPMV- and FnBP-specific serum IgG; however, these titers were significantly lower and more variable than those generated by the intranasal route, and FnBP-specific intestinal IgA was undetectable. Neither the ISCOM matrix nor cholera toxin enhanced these responses. These studies demonstrate for the first time that recombinant plant viruses have potential as mucosal vaccines without the requirement for adjuvant and that the nasal route is most effective for the delivery of these nonreplicating particles.     

Induction of mucosal immunity by intranasal application of a streptococcal surface protein antigen with the cholera toxin B subunit

The level and distribution of isotype-specific antibodies in various secretions and of antibody-secreting cells in corresponding lymphoid organs and tissues were compared in mice immunized with Streptococcus mutans surface protein antigen I/II (AgI/II) conjugated to the cholera toxin B subunit (CTB), given intranasally (i.n.) or intragastrically (i.g.), with or without free cholera toxin (CT) as an adjuvant. Immunization i.n. induced stronger initial antibody responses to AgI/II in both serum and saliva than immunization i.g., but salivary immunoglobulin A (IgA)-specific antibody responses to immunization about 3 months later were not increased relative to total salivary IgA concentrations. Specific antibodies induced by i.n. immunization were as widely distributed in serum, saliva, tracheal wash, gut wash, and vaginal wash as those induced by i.g. immunization. Likewise, specific antibody-secreting cells were generated in the spleen, salivary glands, intestinal lamina propria, and mesenteric and cervical lymph nodes by either route of immunization. The strongest salivary IgA antibody response was induced by AgI/II-CTB conjugate given i.n., but the addition of CT did not further enhance it. However, free CTB could effectively replace CT as an adjuvant in i.n. immunization with unconjugated AgI/II. Booster i.n. immunization with AgI/II plus either free CT or CTB induced stronger recall serum antibody responses than conjugated AgI/II-CTB with or without CT as an adjuvant. Therefore, i.n. immunization with a protein antigen and free or coupled CTB is an effective means of generating IgA antibody responses expressed at several mucosal sites where protective immunity may be beneficial.     

Trichinella spiralis: the effect of oral immunization and the adjuvancy of cholera toxin on the mucosal and systemic immune response of mice

The effect of oral immunization and the adjuvancy of cholera toxin (CT) were examined in mice infected with Trichinella spiralis. The mean of total muscle larvae was reduced by 36% in mice infected with Trichinella larvae in combination with CT. In mice fed soluble, particulate, or soluble/particulate antigens in combination with CT on Days 0, 14, and 21, and challenged with Trichinella larvae on Day 28, there was a significant reduction in adult worm fecundity (50%), worm size (20-30%), and the mean of total muscle larvae (75%) but no apparent effect on the rate of expulsion on Day 6 postchallenge. Following antigen feeding, but prior to challenge with Trichinella (Day 28), the immunoglobulin response was positive in only a small proportion of mice. On Day 6 following challenge with Trichinella larvae, the biliary immunoglobulin response was enhanced approximately 10-fold (P < 0.05) in all antigen-feeding treatments which included CT, compared with nonimmunized controls or antigen-feeding treatments which did not include CT. Similarly, the serum IgG response was enhanced following challenge with Trichinella larvae in treatments fed soluble, particulate, or soluble/particulate antigens with CT. The response was also enhanced in the particulate, but not in soluble or soluble/particulate antigen treatments without CT.     

Nasal immunization of nonhuman primates with simian immunodeficiency virus p55gag and cholera toxin adjuvant induces Th1/Th2 help for virus-specific immune responses in reproductive tissues

Female rhesus macaques were nasally immunized with p55gag (p55) of SIV and cholera toxin as a mucosal adjuvant. Nasal immunization induced Ag-specific IgA and IgG Abs in mucosal secretions (e.g., cervicovaginal secretions, rectal washes, and saliva) and serum. Furthermore, high numbers of p55-specific IgA and IgG Ab-forming cells were induced in mucosal effector sites, i.e., uterine cervix, intestinal lamina propria, and nasal passage. p55-specific CD4+ T cells in both systemic and mucosal compartments expressed IFN-gamma and IL-2 (Th1-type)- as well as IL-5, IL-6, and IL-10 (Th2-type)-specific mRNA. Moreover, p55-specific CTL activity was demonstrated in lymphocytes from blood, tonsils, and other lymphoid tissues. These results show that nasal immunization with SIV p55 with cholera toxin elicits both Th1- and selective Th2-type cytokine responses associated with the induction of SIV-specific mucosal and serum Abs, and CTL activity. These results offer a promise for the development of protective mucosal immunity to SIV.     

Cross-clade p17 peptide recognition by antisera to HGP-30, a 30-amino acid synthetic peptide antigen from HIV type 1 p17

HGP-30, a 30-amino acid synthetic peptide analog of HIV-1SF2 p17 (aa 86-115), was used to immunize both mice and humans. Since the amino acid sequence of HGP-30 is relatively conserved among different HIV-1 strains and clades, experiments were carried out to determine if antisera obtained by immunizing animals and humans can recognize HGP-30-related peptide consensus sequences belonging to different clades. Results show that antisera from mice immunized with HGP-30 can recognize clade B and C and to a lesser degree clade A and E consensus sequences of HIV-1, in addition to recognizing HGP-30 sequence. The cross-clade recognition was higher in mouse sera obtained on day 42 than on day 14 or 28. MPL/SE and Novasomes were better adjuvants than alum in inducing antibodies that showed cross-clade recognition and IgG2a and IgG2b antibody isotypes. Similar cross-clade recognition was observed in several sera from humans immunized with an HGP-30/KLH/alum formulation. The human sera from HGP-30-immunized subjects evaluated for cross-clade recognition of HGP-30 peptides were from subjects whose cells showed significant protection from HIV infection on virus challenge in the hu-PBL-SCID mouse model. These studies suggest that HGP-30 may be useful as a candidate vaccine antigen for populations in countries with prevalence of different HIV clades.     

Oral administration of polymer-grafted starch microparticles activates gut-associated lymphocytes and primes mice for a subsequent systemic antigen challenge

The mucosal and systemic humoral immune systems can function essentially independent of each other, responding to mucosal and parenteral antigens, respectively. Nevertheless, antigen administered by one route can modify responsiveness to subsequent immunization by an alternate route. Here we demonstrated, in mice, in addition to stimulating rapid and robust sera antibody responses, intragastric (i.g.) immunization with human serum albumin (HSA)-containing starch microparticles (MP) grafted with 3-(triethoxysilyl)-propyl-terminated polydimethylsiloxane (TS-PDMS) primed for enhanced specific sera IgG following a parenteral antigen boost. After as little as one i.g. immunization with microentrapped, but not with soluble, HSA antigen-specific proliferation and antibody secretion were detected in Peyer's patches (PP); this activity peaked after three i.g. MP immunizations. We observed a progressive dissemination of antigen-specific lymphocyte reactivity from PP to splenic tissue following oral MP immunization. Similarly, we observed a shift in HSA-specific antibody-secreting cells from PP and mesenteric lymph nodes to splenic tissue following i.g. MP immunization. We also demonstrated that oral immunization with microentrapped, but not with soluble HSA, resulted in enhanced numbers of spontaneous Th2-cytokine secreting lymphocytes which disseminated from mucosal to systemic lymphoid compartments. This observation coincided with our findings that HSA-specific sera IgG1 responses in animals given HSA in MP were significantly higher than those detected in the sera of mice given soluble HSA i.g., both before and after parenteral antigen challenge. These findings suggest that orally-administered TS-PDMS-grafted MP, by stimulating elements of the mucosal immune system, are a valuable addition to mucosal and systemic vaccine delivery systems.     

A nontoxic adjuvant for mucosal immunity to pneumococcal surface protein A

In this study, we demonstrated that pneumococcal surface protein A (PspA) nasally administered with a nontoxic A subunit mutant of cholera toxin (mCT) S61F elicited a protective immune response. Immunization with PspA and mCT elicited higher levels of PspA-specific IgG and IgA Abs in serum and of IgG and IgA anti-PspA Ab-forming cells in spleens, cervical lymph nodes (CLN), and lung tissue when compared to nonimmunized mice. Furthermore, significant PspA-specific IgA Abs were induced in saliva and nasal secretions. These responses were dependent on the use of mCT as a mucosal adjuvant. The PspA-specific Ab responses induced by mCT S61F were comparable with those induced by native CT (nCT). Analysis of cytokine responses showed that nasal PspA plus mCT S61F enhanced the induction of PspA-specific CD4+ T cells producing IL-4 but not IFN-gamma in CLN at both the protein and mRNA levels. Importantly, significant numbers of mice intranasally immunized with PspA plus mCT S61F were protected from lethal challenge with capsular serotype 3 Streptococcus pneumoniae A66. These results show that intranasal administration of PspA together with mCT S61F is an effective mucosal vaccine against pneumococcal infection and induces CD4+ Th2-type cells, which provide help for both mucosal and systemic Ab responses.     

Nasal immunization with group B streptococci can induce high levels of specific IgA antibodies in cervicovaginal secretions of mice

We have studied the cervicovaginal antibody responses in mice, by ELISA, following mucosal immunizations with group B streptococci (GBS) serotype III/R4. Immunizations were carried out either: (1) rectally with GBS alone; (2) rectally with GBS plus cholera toxin (CT); (3) nasally with GBS alone; (4) nasally with GBS+CT; or (5) nasally under general anesthesia with GBS+CT. Nasal immunizations with GBS alone led to at least tenfold higher levels of specific IgA-antibodies to GBS in cervicovaginal secretions than with any other immunization. These mucosal antibody levels were higher than after rectal immunizations, and 2-17 times higher than the corresponding IgA antibody levels in sera. Markedly lower cervicovaginal antibody levels were found in mice which had received GBS together with CT as a mucosal adjuvant than in mice immunized by the same routes with GBS alone. Our observations indicate that a nasal vaccine consisting of GBS might induce sufficient antibody levels to protect against genital colonization of these bacteria.     

Evidence for serum immunoglobulin G (IgG) antibody responses in Macaca fascicularis identified by monoclonal antibodies to human IgG subclasses

This investigation determined the capacity of murine monoclonal antibodies directed to human immunoglobulin G (IgG) subclasses to identify molecules with conserved epitopes in the serum of the nonhuman primate, Macaca fascicularis. We subsequently utilized this cross-reactivity to document the characteristics of IgG subclass antibody responses in M. fascicularis to parenteral immunization with intact oral microorganisms, antigens from oral microorganisms, and finally a defined protein toxin, tetanus toxoid. The IgG response in nonhuman primates immunized with tetanus toxoid showed a 40-fold and 110-fold increase after primary and secondary immunizations, respectively. The major IgG subclass responses were IgG1 and IgG3, with little, though significant, responses in the IgG4 and IgG2 subclasses. Seventy-five to 94% of the natural IgG antibody in nonhuman primate sera to Porphyromonas gingivalis, Prevotella intermedia and Campylobacter rectus was IgG1. IgG2 and IgG3 predominated to Bacteroides fragilis, IgG4 to Actinomyces viscosus and an equal distribution among the subclasses was noted in response to Fusobacterium nucleatum. Parenteral immunization of nonhuman primates with intact P. gingivalis elicited primarily IgG3 and IgG4, while the post-immunization IgG response to P. intermedia was largely IgG1. Nonhuman primates were also parenterally immunized with cell envelope antigens of P. gingivalis, P. intermedia, or a combination of cell envelope antigen from C. rectus and F. nucleatum and cell wall antigens of A. viscosus. The greatest IgG antibody response seen post-immunization was reactive with anti-human IgG1 for all of these antigens except to C. rectus which bound nonhuman primate antibody reactive with anti-human IgG2. It appears that the bacteria and their products exhibit unique differences in their induction of serum IgG subclass antibody responses. The characteristics of their immunogenicity as detected by the nonhuman primate may contribute to the ability of the immune responses to effectively interact with these pathogens.