Epitopes of group A streptococcal M protein that evoke cross-protective local immune responses.

The present studies were undertaken to identify conserved epitopes of group A streptococcal M proteins that evoke cross-protective mucosal immune responses. Two synthetic peptides copying conserved regions of type 5 M protein, designated SM5(235-264)C and SM5(265-291)C, were covalently linked to carrier molecules and their immunogenicity was tested in laboratory animals. Rabbit antisera against both peptides cross-reacted with multiple serotypes of group A streptococci, indicating that the peptides contained broadly cross-reactive, surface exposed M protein epitopes. Serum antipeptide antibodies adsorbed to the surface of heterologous type 24 streptococci passively protected mice against intranasal challenge infections. Mice that were actively immunized intranasally with each synthetic peptide covalently linked to the B subunit of cholera toxin were protected against colonization and death after intranasal challenge infections with type 24 streptococci in the absence of serum opsonic antibodies. These data confirm and extend previous observations that conserved M protein epitopes evoke cross-protective local immunity and may serve as the basis for broadly cross-protective M protein vaccines.     

Protective immunity induced by an intranasal multivalent vaccine comprising 10 Lactococcus lactis strains expressing highly prevalent M‐protein antigens derived from Group A Streptococcus

Streptococcus pyogenes (group A Streptococcus) causes diseases ranging from mild pharyngitis to severe invasive infections. The N‐terminal fragment of streptococcal M protein elicits protective antibodies and is an attractive vaccine target. However, this N‐ terminal fragment is hypervariable: there are more than 200 different M types. In this study, an intranasal live bacterial vaccine comprising 10 strains of Lactococcus lactis, each expressing one N‐terminal fragment of M protein, has been developed. Live bacterial‐vectored vaccines cost less to manufacture because the processes involved are less complex than those required for production of protein subunit vaccines. Moreover, intranasal administration does not require syringes or specialized personnel. Evaluation of individual vaccine types (M1, M2, M3, M4, M6, M9, M12, M22, M28 and M77) showed that most of them protected mice against challenge with virulent S. pyogenes. All 10 strains combined in a 10‐valent vaccine (M×10) induced serum and bronchoalveolar lavage IgG titers that ranged from three‐ to 10‐fold those of unimmunized mice. After intranasal challenge with M28 streptococci, survival of M×10‐immunized mice was significantly higher than that of unimmunized mice. In contrast, when mice were challenged with M75 streptococci, survival of M×10‐immunized mice did not differ significantly from that of unimmunized mice. Mx‐10 immunized mice had significantly less S. pyogenes in oropharyngeal washes and developed less severe disease symptoms after challenge than did unimmunized mice. Our L. lactis‐based vaccine may provide an alternative solution to development of broadly protective group A streptococcal vaccines.

     

Antibody Response and Protection Induced by Immunization with Smooth and Rough Strains in Experimental Salmonellosis

The antibody response of mice to a smooth strain of Salmonella typhimurium was shown previously to be extremely rapid and potent. As measured by the complement-mediated bactericidal reaction, it was also found to be highly specific as well as reproducible. Experiments which studied the effects of antigen type (live or heat-killed), antigen dose, and the route of immunization indicated that the most rapid and highest antibody response was achieved with live, smooth organisms injected by the intraperitoneal route. Living vaccines of rough strains of either S. typhimurium or S. enteritidis induced antibodies directed against the corresponding smooth organisms. The response to the rough strains was apparently due to antibody production rather than to the simple release of preformed natural antibody. The duration of protection conferred by the rough strain vaccines was closely correlated with the endotoxic content of the immunizing strain. Smooth heat-killed vaccines and a rough live vaccine protected against homologous but not heterologous challenge. In contrast, immunization with a smooth live vaccine protected mice against both homologous and heterologous challenge infections. Protection was not due to a local effect in the peritoneal cavity, since mice were also protected against subcutaneous challenge. The secondary antibody response, induced in immunized animals by the virulent challenge infection, was demonstrated to be rapid and potent, and hence a factor to be considered in protection.     

Age Dependence of Immunity Induced by a Candidate Universal Influenza Vaccine in Mice

Influenza has a major impact on the elderly due to increased susceptibility to infection with age and poor response to current vaccines. We have studied universal influenza vaccine candidates based on influenza A nucleoprotein and matrix 2 (A/NP+M2). Long-lasting protection against influenza virus strains of divergent subtypes is induced, especially with mucosal immunization. Here, we tested universal vaccination in BALB/c mice of different ages. Vaccination used intramuscular DNA priming to A/NP+M2 followed by intranasal (i.n.) boosting with recombinant adenoviruses (rAd) expressing the same antigens, or only A/NP+M2-rAd given i.n. Antigen-specific systemic antibody responses were induced in young, middle-aged, and elderly mice (2, 11–17, and 20 months old, respectively), but decreased with age. Antibody responses in bronchoalveolar lavage (BAL) were detected only in young mice. Antigen-specific T cell responses were seen in young and middle-aged but not elderly mice. A/NP+M2 vaccination by the two regimens above protected against stringent challenge in young and middle-aged mice, but not in elderly mice. However, mice vaccinated with A/NP-rAd or A/M2-rAd during their youth were partially protected against challenge 16 months later when they were elderly. In addition, a regimen of two doses of A/NP+M2-rAd given i.n. one month apart beginning in old age protected elderly mice against stringent challenge. This study highlights the potential benefit of cross-protective vaccines through middle age, and suggests that their performance might be enhanced in elderly individuals who had been exposed to influenza antigens early in life, as most humans have been, or by a two-dose rAd regimen given later in life.     

Mucosal delivery of inactivated influenza vaccine induces B-cell-dependent heterosubtypic cross-protection against lethal influenza A H5N1 virus infection

Influenza vaccines that induce greater cross-reactive or heterosubtypic immunity (Het-I) may overcome limitations in vaccine efficacy imposed by the antigenic variability of influenza A viruses. We have compared mucosal versus traditional parenteral administration of inactivated influenza vaccine for the ability to induce Het-I in BALB/c mice and evaluated a modified Escherichia coli heat-labile enterotoxin adjuvant, LT(R192G), for augmentation of Het-I. Mice that received three intranasal (i.n.) immunizations of H3N2 vaccine in the presence of LT(R192G) were completely protected against lethal challenge with a highly pathogenic human H5N1 virus and had nasal and lung viral titers that were at least 2,500-fold lower than those of control mice receiving LT(R192G) alone. In contrast, mice that received three vaccinations of H3N2 vaccine subcutaneously in the presence or absence of LT(R192G) or incomplete Freund's adjuvant were not protected against lethal challenge and had no significant reductions in tissue virus titers observed on day 5 post-H5N1 virus challenge. Mice that were i.n. administered H3N2 vaccine alone, without LT(R192G), displayed partial protection against heterosubtypic challenge. The immune mediators of Het-I were investigated. The functional role of B and CD8+ T cells in Het-I were evaluated by using gene-targeted B-cell (IgH-6(-/-))- or beta2-microglobulin (beta2m(-/-))-deficient mice, respectively. beta2m(-/-) but not IgH-6(-/-) vaccinated mice were protected by Het-I and survived a lethal infection with H5N1, suggesting that B cells, but not CD8+ T cells, were vital for protection of mice against heterosubtypic challenge. Nevertheless, CD8+ T cells contributed to viral clearance in the lungs and brain tissues of heterotypically immune mice. Mucosal but not parenteral vaccination induced subtype cross-reactive lung immunoglobulin G (IgG), IgA, and serum IgG anti-hemagglutinin antibodies, suggesting the presence of a common cross-reactive epitope in the hemagglutinins of H3 and H5. These results suggest a strategy of mucosal vaccination that stimulates cross-protection against multiple influenza virus subtypes, including viruses with pandemic potential.     

Epitope specific immunity elicited by a synthetic streptococcal antigen without carrier or adjuvant

A polypeptide fragment of type 24 streptococcal M protein (pep M24) has been shown to raise protective anti-streptococcal antibodies in rabbits and humans when administered with adjuvants. More recently, such protective antibodies were shown to be evoked by a synthesized 35-residue sub-peptide fragment (S-CB7 synthetic cyanogen bromide fragment 7) of pep M24. We now show that the weak pep M24 immunogen induces high titers of long lasting antibodies when associated with murabutide, a synthetic derivative of MDP (NAcMur-L-Ala-D-Glnn-butyl-ester) which is currently undergoing clinical trials. We demonstrate also that the polymerized synthetic S-CB7 administered without adjuvant or carrier evokes a strong epitope specific, protective immune response in mice primed with the parent pep M24. A booster dose of polymerized S-CB7 induced antibodies directed specifically against the S-CB7 structure whereas a booster dose of pep M24 evoked antibodies recognizing additional determinants of the whole pep M24 molecule.     

An oral vaccine based on U-Omp19 induces protection against B. abortus mucosal challenge by inducing an adaptive IL-17 immune response in mice

As Brucella infections occur mainly through mucosal surfaces, the development of mucosal administered vaccines could be radical for the control of brucellosis. In this work we evaluated the potential of Brucella abortus 19 kDa outer membrane protein (U-Omp19) as an edible subunit vaccine against brucellosis. We investigated the protective immune response elicited against oral B. abortus infection after vaccination of mice with leaves from transgenic plants expressing U-Omp19; or with plant-made or E. coli-made purified U-Omp19. All tested U-Omp19 formulations induced protection against Brucella when orally administered without the need of adjuvants. U-Omp19 also induced protection against a systemic challenge when parenterally administered. This built-in adjuvant ability of U-Omp19 was independent of TLR4 and could be explained at least in part by its capability to activate dendritic cells in vivo. While unadjuvanted U-Omp19 intraperitoneally administered induced a specific Th1 response, following U-Omp19 oral delivery a mixed specific Th1-Th17 response was induced. Depletion of CD4(+) T cells in mice orally vaccinated with U-Omp19 resulted in a loss of the elicited protection, indicating that this cell type mediates immune protection. The role of IL-17 against Brucella infection has never been explored. In this study, we determined that if IL-17A was neutralized in vivo during the challenge period, the mucosal U-Omp19 vaccine did not confer mucosal protection. On the contrary, IL-17A neutralization during the infection did not influence at all the subsistence and growth of this bacterium in PBS-immunized mice. All together, our results indicate that an oral unadjuvanted vaccine based on U-Omp19 induces protection against a mucosal challenge with Brucella abortus by inducing an adaptive IL-17 immune response. They also indicate different and important new aspects i) IL-17 does not contribute to reduce the bacterial burden in non vaccinated mice and ii) IL-17 plays a central role in vaccine mediated anti-Brucella mucosal immunity.     

Sublingual immunization with M2-based vaccine induces broad protective immunity against influenza

Background

The ectodomain of matrix protein 2 (M2e) of influenza A virus is a rationale target antigen candidate for the development of a universal vaccine against influenza as M2e undergoes little sequence variation amongst human influenza A strains. Vaccine-induced M2e-specific antibodies (Abs) have been shown to display significant cross-protective activity in animal models. M2e-based vaccine constructs have been shown to be more protective when administered by the intranasal (i.n.) route than after parenteral injection. However, i.n. administration of vaccines poses rare but serious safety issues associated with retrograde passage of inhaled antigens and adjuvants through the olfactory epithelium. In this study, we examined whether the sublingual (s.l.) route could serve as a safe and effective alternative mucosal delivery route for administering a prototype M2e-based vaccine. The mechanism whereby s.l. immunization with M2e vaccine candidate induces broad protection against infection with different influenza virus subtypes was explored.

Methods and Results

A recombinant M2 protein with three tandem copies of the M2e (3M2eC) was expressed in Escherichia coli. Parenteral immunizations of mice with 3M2eC induced high levels of M2e-specific serum Abs but failed to provide complete protection against lethal challenge with influenza virus. In contrast, s.l. immunization with 3M2eC was superior for inducing protection in mice. In the latter animals, protection was associated with specific Ab responses in the lungs.

Conclusions

The results demonstrate that s.l. immunization with 3M2eC vaccine induced airway mucosal immune responses along with broad cross-protective immunity to influenza. These findings may contribute to the understanding of the M2-based vaccine approach to control epidemic and pandemic influenza infections.     

Biochemical and biological properties of the binding of human fibrinogen to M protein in group A streptococci.

Fibrinogen is known to bind to group A streptococci and precipitate with extracts containing streptococcal M protein. We have previously shown that the binding of fibrinogen to M-positive streptococci prevents opsonization by complement and protects that organism from phagocytosis in nonimmune blood. In the present study, we used 3H-labeled fibrinogen, a highly purified peptide fragment of type 24 M protein (pep M24), and anti-pep M sera to show that fibrinogen binds to M-positive streptococci with high affinity (dissociation constants, 1 to 5 nM); occupation of the high-affinity binding sites suffices to protect the organism from phagocytosis; proteolytic treatments that remove M protein from streptococcal cells abolish binding; binding is competitively inhibited by anti-pep M sera; pep M24 precipitates fibrinogen; and binding to type 24 cells is inhibited by pep M24. We conclude that M protein is the cell surface structure principally responsible for binding fibrinogen on the surface of M-positive streptococci and that this binding contributes to the known antiopsonic property of M proteins.     

Protective and nonprotective epitopes of chemically synthesized peptides of the NH2-terminal region of type 6 streptococcal M protein

The protective immunogenicity of chemically synthesized copies of the NH2-terminal region of type 6 streptococcal M protein was investigated. Four overlapping peptides were synthesized by copying residues 1-20, 10-20, 12-31, and 22-31. Rabbit antisera raised against whole cells of type 6 streptococci reacted at high dilutions (1/12,800 to 1/51,200) with S-M6(1-20) and S-M6(10-20), and at low dilutions (1/100-1/800) with S-M6(12-31) and S-M6(22-31), indicating that the NH2-terminal region of type 6 M protein bears immunodominant epitopes. When covalently linked to tetanus toxoid and emulsified in complete Freund's adjuvant, the synthetic peptides S-M6(1-20), S-M6(10-20), and S-M6(12-31), but not S-M6(22-31), evoked type-specific opsonic antibodies against type 6 streptococci. Although the immune sera reacted in low dilutions by enzyme linked immunoabsorbent assay (ELISA) with the heterologous M protein polypeptides pep M5, pep M19, and pep M24, they failed to opsonize the streptococci from which these M protein polypeptides were derived. Each of the immune sera reacted in high dilution by ELISA with the respective immunizing peptides. All except those against S-M6(22-31) also reacted with pep M6. None of the immune sera reacted with human cardiac tissue by immunofluorescence or with muscle myosin by ELISA. The pattern of the inhibition of opsonization by each of the synthetic peptides of each of the immune sera indicates the presence of at least three protective epitopes in the NH2-terminal region of type 6 M protein. Our results indicate that the NH2-terminal region of type 6 M protein contains both protective and nonprotective epitopes, and chemically synthesized copies of this region lack cardiac tissue cross-reactive epitopes. These studies hold promise for the development of safe and effective vaccines against group A streptococci, especially against the strains giving rise to rheumatic fever and rheumatic heart disease.     

An oligosaccharide-tetanus toxoid conjugate vaccine against type III group B Streptococcus

We have developed an oligosaccharide-tetanus toxoid conjugate vaccine against type III group B Streptococcus. Purified group B streptococcal type III capsular polysaccharide was depolymerized by enzymatic digestion using endo-beta-galactosidase produced by Citrobacter freundii. Following enzymatic digestion, oligosaccharides were fractionated by gel filtration chromatography on Sephadex G-75. An oligosaccharide pool of average Mr = 14,500 (corresponding to 13.6 repeating units of the type III polysaccharide) was used for conjugation to tetanus toxoid. Tetanus toxoid was covalently coupled via a synthetic spacer molecule to the reducing end of the oligosaccharide by reductive amination. The oligosaccharide-tetanus toxoid conjugate elicited type III-specific anticapsular antibodies (measured in enzyme-linked immunosorbent assay) in three out of three rabbits whereas the unconjugated native type III polysaccharide was nonimmunogenic. Antiserum from rabbits vaccinated with the oligosaccharide-protein conjugate protected mice against lethal challenge with live group B streptococci (16 out of 16 mice survived) and opsonized group B streptococci for phagocytosis in vitro. No protection was conferred by preimmune serum nor by serum from rabbits vaccinated with unconjugated native type III polysaccharide. An oligosaccharide-protein conjugate vaccine of this design may prove to be an effective immunogen for protection against group B streptococcal infection in humans. In addition, the approach to vaccine design utilized in these studies will facilitate further definition of the structural parameters that determine immune response to glycoconjugate vaccines.     

Domains of group A streptococcal M protein that confer resistance to phagocytosis, opsonization and protection: implications for vaccine development

Streptococcus pyogenes (group A streptococcus) colonizes skin and throat tissues resulting in a range of benign and serious human diseases. Opsonization and phagocytosis are important defence mechanisms employed by the host to destroy group A streptococci. Antisera against the cell-surface M protein, of which over 150 different types have been identified, are opsonic and contribute to disease protection. In this issue of Molecular Microbiology, Sandin and colleagues have comprehensively analysed the regions of M5 protein that contribute to phagocytosis resistance and opsonization. Human plasma proteins bound to M5 protein B- and C-repeats were shown to block opsonization, an observation that needs to be carefully considered for the development of M protein-derived vaccines. While safe and efficacious human group A streptococcal vaccines are not commercially available, candidate M protein-derived vaccines have shown promise in murine vaccine models and a recent phase 1 human clinical trial.     

Cellular and biochemical responses of human T lymphocytes stimulated with streptococcal M proteins

Purified group A streptococcal M proteins, pep M5 and pep M6, bearing heart cross-reactive epitopes were compared with pep M24, which lacks such epitopes, in their ability to induce functional differentiation of human T lymphocytes. Lymphocytes activated by pep M5 and pep M6 demonstrated cytotoxic activity against cultured heart cells, whereas pep M24-activated cells differentiated into suppressor T cells, which specifically blocked cytotoxic T lymphocytes against cultured human myocardial cells and not NK cell activity against K562 cells. Pep M5 and not pep M24 induced an increase in the number of CD4, 4B4, helper/inducer T cells. In addition, these M proteins appear to induce different biochemical changes in T lymphocytes. Both pep M5 and pep M24 induced the phosphorylation of a 35-kDa cytoplasmic protein; however, only pep M5 induced the phosphorylation of a 28-kDa membrane protein, primarily in CD4 T cells. These data indicate that the virulent M protein Ag of group A streptococci may exert their effect on the human immune system via different mechanisms. Determining these mechanisms and the biochemical pathways involved in T cell differentiation triggered by these Ag may be important in understanding the pathogenesis of post-streptococcal diseases.     

Mechanisms of mucosal and parenteral tuberculosis vaccinations: adenoviral-based mucosal immunization preferentially elicits sustained accumulation of immune protective CD4 and CD8 T cells within the airway lumen

The mechanisms underlying better immune protection by mucosal vaccination have remained poorly understood. In our current study we have investigated the mechanisms by which respiratory virus-mediated mucosal vaccination provides remarkably better immune protection against pulmonary tuberculosis than parenteral vaccination. A recombinant adenovirus-based tuberculosis (TB) vaccine expressing Mycobacterium tuberculosis Ag85A (AdAg85A) was administered either intranasally (i.n.) or i.m. to mice, and Ag-specific CD4 and CD8 T cell responses, including frequency, IFN-gamma production, and CTL, were examined in the spleen, lung interstitium, and airway lumen. Although i.m. immunization with AdAg85A led to activation of T cells, particularly CD8 T cells, in the spleen and, to a lesser extent, in the lung interstitium, it failed to elicit any T cell response in the airway lumen. In contrast, although i.n. immunization failed to effectively activate T cells in the spleen, it uniquely elicited higher numbers of Ag-specific CD4 and CD8 T cells in the airway lumen that were capable of IFN-gamma production and cytolytic activities, as assessed by an intratracheal in vivo CTL assay. These airway luminal T cells of i.n. immunized mice or splenic T cells of i.m. immunized mice, upon transfer locally to the lungs of naive SCID mice, conferred immune protection against M. tuberculosis challenge. Our study has demonstrated that the airway luminal T cell population plays an important role in immune protection against pulmonary TB, thus providing mechanistic insights into the superior immune protection conferred by respiratory mucosal TB vaccination.     

A recombinant multivalent combination vaccine protects against Chlamydia and genital herpes

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-gamma 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.     

A viral vaccine vector that expresses foreign genes in lymph nodes and protects against mucosal challenge.

A candidate live-virus vaccine strain of Venezuelan equine encephalitis virus (VEE) was configured as a replication-competent vector for in vivo expression of heterologous immunogens. Three features of VEE recommend it for use as a vaccine vector. (i) Most human and animal populations are not already immune to VEE, so preexisting immunity to the vector would not limit expression of the heterologous antigen. (ii) VEE replicates first in local lymphoid tissue, a site favoring the induction of an effective immune response. (iii) Parenteral immunization of rodents and humans with live, attenuated VEE vaccines protects against mucosal challenge, suggesting that VEE vaccine vectors might be used successfully to protect against mucosal pathogens. Upon subcutaneous (s.c.) inoculation into the footpad of mice, a VEE vector containing the complete influenza virus hemagglutinin (HA) gene expressed HA in the draining lymph node and induced anti-HA immunoglobulin G (IgG) and IgA serum antibodies, the levels of which could be increased by s.c. booster inoculation. When immunized mice were challenged intranasally with a virulent strain of influenza virus, replication of challenge virus in their lungs was restricted, and they were completely protected from signs of disease. Significant reduction of influenza virus replication in the nasal epithelia of HA vector-immunized mice suggested an effective immunity at the mucosal surface. VEE vaccine vectors represent an alternative vaccination strategy when killed or subunit vaccines are ineffective or when the use of a live attenuated vaccine might be unsafe.     

Do we need nasal vaccines against COVID 19 to suppress the transmission of infections?

Covid-19 vaccines have within the first year prevented about 14 million deaths but did not induce a strong mucosal immune response. Data from US, UK, Singapore and Israel showed a variable and mostly modest effects of vaccination on virus excretion during breakthrough infections. Contact studies showed decreased transmission of infection from vaccinated index cases, but the effect varied according to dominant virus type, with study type and the nature of the contact group and diminished with time after vaccination. Some researchers suspect that it is unlikely to stop the pandemic with injected vaccines alone. Promising animal experiments were conducted with mucosal vaccines. Mice nasally immunized with a chimpanzee adenovirus vector mounted a mucosal immune response, were protected against viral challenge after a single vaccine dose and suppressed nasal replication of the challenge virus. Phage T4 expressing SARS-CoV-2 spike and nucleocapsid induced a sterilizing lung immunity in nasally vaccinated mice. Also hamsters intranasally immunized with the prefusion-stabilized spike protein showed no infectious virus in nasal turbinates upon challenge. Other studies showed that intranasal vaccination with an adenovirus vaccine reduced but did not eliminated viral transmission from infected to naïve hamsters. Intranasal vaccination of rhesus macaques with adenovirus vaccines also substantially reduced or even suppressed viral replication in the upper and lower respiratory tract. Human data on mucosal SARS-CoV-2 vaccines are so far limited to safety and immunogenicity studies. Aerosolized adenovirus vaccines given either as a booster or as primary immunization were safe and induced similar or superior immune response than injected vaccines while an aerosolized influenza vectored vaccine induced only a weak humoral and cellular immune response. Overall 100 mucosal SARS-CoV-2 vaccines are in development and 20 are in clinical trials. First human trials demonstrate that this will not be an easy task.     

A neonatal Fc receptor-targeted mucosal vaccine strategy effectively induces HIV-1 antigen-specific immunity to genital infection

Strategies to prevent the sexual transmission of HIV include vaccines that elicit durable, protective mucosal immune responses. A key to effective mucosal immunity is the capacity for antigens administered locally to cross epithelial barriers. Given the role of neonatal Fc receptor (FcRn) in transferring IgG across polarized epithelial cells which line mucosal surfaces, FcRn might be useful for delivering HIV vaccine antigens across mucosal epithelial barriers to the underlying antigen-presenting cells. Chimeric proteins composed of HIV Gag (p24) fused to the Fc region of IgG (Gag-Fc) bind efficiently to airway mucosa and are transported across this epithelial surface. Mice immunized intranasally with Gag-Fc plus CpG adjuvant developed local and systemic immunity, including durable B and T cell memory. Gag-specific immunity was sufficiently potent to protect against an intravaginal challenge with recombinant vaccinia virus expressing the HIV Gag protein. Intranasal administration of a Gag-Fc/CpG vaccine protected at a distal mucosal site. Our data suggest that targeting of FcRn with chimeric immunogens may be an important strategy for mucosal immunization and should be considered a new approach for preventive HIV vaccines.     

Antibody responses elicited by a polyvalent vaccine containing synthetic diphtheric, streptococcal and hepatitis peptides coupled to the same carrier

Three synthetic peptides copying fragments of the diphtheria toxin, the M protein of the streptococcus type 24 and the hepatitis B virus surface antigen (HBs) have been conjugated together to the tetanus toxoid. This polyvalent vaccine has been administered to mice. High antibody titers were obtained against the three antigens. No cross-reactivity could be observed between them as demonstrated by the ability of each peptide to inhibit only the antibodies against the natural M protein and the synthetic M protein peptide indicated that the avidity of the antibodies raised against a monovalent streptococcal vaccine were identical to those raised following injection of the polyvalent vaccine. Antibodies raised against the polyvalent streptococcal vaccine were also protective as shown by opsonophagocytic assays.     

Mucosal luminal manipulation of T cell geography switches on protective efficacy by otherwise ineffective parenteral genetic immunization

Genetic immunization holds great promise for future vaccination against mucosal infectious diseases. However, parenteral genetic immunization is ineffective in control of mucosal intracellular infections, and the underlying mechanisms have remained unclear. By using a model of parenteral i.m. genetic immunization and pulmonary tuberculosis (TB), we have investigated the mechanisms that determine the failure and success of parenteral genetic immunization. We found that lack of protection from pulmonary Mycobacterium tuberculosis (M.tb) challenge by i.m. immunization with a recombinant adenovirus-vectored tuberculosis vaccine was linked to the absence of M.tb Ag-specific T cells within the airway lumen before M.tb challenge despite potent T cell activation in the systemic compartments. Furthermore, pulmonary mycobacterial challenge failed to recruit CD8 T cells into the airway lumen of i.m. immunized mice. Such defect in T cell recruitment, intra-airway CTL, and immune protection was restored by creating acute inflammation in the airway with inflammatory agonists such as virus. However, the Ag-specific T cells recruited as such were not retained in the airway lumen, resulting in a loss of protection. In comparison, airway exposure to low doses of soluble M.tb Ags not only recruited but retained Ag-specific CD8 T cells in the airway lumen over time that provided robust protection against M.tb challenge. Thus, our study reveals that mucosal protection by parenteral immunization is critically determined by T cell geography, i.e., whether Ag-specific T cells are within or outside of the mucosal lumen and presents a feasible solution to empower parenteral immunization strategies against mucosal infectious diseases.