Evaluation of human immunodeficiency virus type 1 subtype C gag, pol, and gagpol DNA and alphavirus replicon vaccines

The worldwide HIV-1 vaccine research endeavor is focused increasingly on subtype C, which is now the predominant strain of the present HIV/AIDS epidemic. Expression cassettes of HIV-1 subtype C gag, pol and versions of gagpol fusion cassettes were constructed and evaluated for their relative abilities to induce cellular immune responses in mice. Animals were vaccinated with DNA or alphavirus replicon particle-based vaccines and cellular immune responses were measured by flow cytometry. Five new major histocompatibility complex (MHC) class I-restricted T cell epitopes in subtype C Gag and Pol were identified. Although two CD8(+) T cell epitopes within Gag were immunodominant in BALB/c and CB6F1 mice, the overall breadth of the T cell responses in mice immunized with plasmids or recombinant alphavirus replicon particles encoding gagpol fusion genes was improved over single antigen genes (i.e. gag or pol alone). The patterns of epitope dominance were consistent among mice although there were variations observed between different animals in the relative contributions of the various epitopes to the total response. These data are consistent with observations in non-human primates (Otten GR, Schaefer M, Doe B, Liu H, Magede JZ, Donnelly J, et al. Potent immunogenicity of an HIV-1 gag-pol fusion DNA vaccine delivered by in vivo electroporation. Vaccine 2005, in press) and support a subtype C in-frame gagpol fusion gene vaccine.     

A chimeric alphavirus RNA replicon gene-based vaccine for human parainfluenza virus type 3 induces protective immunity against intranasal virus challenge

Parainfluenza virus type 3 (PIV3) infections continue to be a significant health risk for infants, young children, and immunocompromised adults. We describe a gene-based vaccine strategy against PIV3 using replication-defective alphavirus vectors. These RNA replicon vectors, delivered as virus-like particles and expressing the PIV3 hemagglutinin-neuraminidase glycoprotein, were shown to be highly immunogenic in mice and hamsters, inducing PIV3-specific neutralizing antibody responses. Importantly, the replicon particle-based vaccine administered intramuscularly or intranasally protected against mucosal PIV3 challenge in hamsters, preventing virus replication in both nasal turbinates and lungs. These data suggest that the alphavirus replicon platform can be useful for a PIV3 vaccine and possibly other respiratory viruses.     

Construction and cellular immune response induction of HA-based alphavirus replicon vaccines against human-avian influenza (H5N1)

Several approaches are being taken worldwide to develop vaccines against H5N1 viruses; most of them, however, pose both practical and immunological challenges. One potential strategy for improving the immunogenicity of vaccines involves the use of alphavirus replicons and VP22, a herpes simplex type 1 (HSV-1) protein. In this study, we analysed the antigenic peptides and homogeneity of the HA sequences (human isolates of the H5N1 subtype, from 1997 to 2003) and explored a novel alphavirus replicon system of VP22 fused with HA, to assess whether the immunogenicity of an HA-based replicon vaccine could be induced and augmented via fusion with VP22. Further, replicon particles expressing VP22, and enhanced green fluorescent protein (EGFP) were individually used as controls. Cellular immune responses in mice immunised with replicons were evaluated by identifying specific intracellular cytokine production with flow cytometry (FCM). Animal-based experimentation indicated that both the IL-4 expression of CD4⁺ T cells and the IFN-γ expression of CD8⁺ T cells were significantly increased in mice immunised with VPR-HA and VPR-VP22/HA. A dose titration effect vis-à-vis both IL-4 expression and IFN-γ expression were observed in VPR-HA- and VPR-VP22/HA-vaccinated mice. Our results revealed that both VPR-VP22/HA and VPR-HA replicon particles presented a promising approach for developing vaccines against human-avian influenza, and VP22 could enhance the immunogenicity of the HA antigens to which it is fused.     

Protective immunity induced by concurrent intradermal injection of porcine circovirus type 2 and Mycoplasma hyopneumoniae inactivated vaccines in pigs

Vaccines against porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (Mhp) are routinely used by intramuscular injection. However, since intramuscular vaccination causes stress and increases the risk of cross-contamination among pigs, research on intradermal vaccination is currently being actively conducted. This study was designed to evaluate the efficacy of intradermally administered inactivated vaccines against PCV2 and Mhp in pigs. Three-week-old specific pathogen-free pigs were divided into three groups (5 pigs per group). Pigs in the two groups were intradermally vaccinated with the PCV2 or Mhp vaccine using a needle-free injector. Pigs in the third group were kept as nonvaccinated controls. At 21 days post-vaccination, pigs in one of these vaccinated groups and the nonvaccinated group were intranasally challenged with PCV2b and Mhp, while the other vaccinated group pigs were maintained as vaccine controls. Vaccine efficacy was evaluated by observing weight gain, pathogen load, pathological changes, and humoral or cellular immune responses. As a result, vaccinated pigs revealed significantly higher body weight gain, with lower clinical scores. Vaccinated pigs also showed higher antibody responses but lower PCV2b or Mhp loads in sera, nasal swabs, or lungs than nonvaccinated pigs. Intriguingly, vaccinated pigs upregulated cytotoxic T cells (CTLs), helper T type 1 cells (Th1 cells), and helper T type 17 cells (Th17 cells) after immunization and showed significantly higher levels of CTLs, Th1 and Th17 cells at 14 days post-challenge than nonvaccinated and challenged pigs. This study demonstrated that protective immune responses against PCV2 and Mhp could be efficiently induced in pigs using a relatively small volume of intradermal vaccines, probably due to effective antigen delivery to antigen-presenting cells in the dermis.     

Ⅰ型干扰素受体与慢性病毒性肝炎的相关性

在慢性病毒性肝炎治疗中,IFN通过与细胞表面特异性受体结合发挥其抗病毒和免疫调节效应的作用.作为关键环节的IFN受体与慢性病毒性肝炎的易感性和治疗效果密切相关,此文对IFN受体与慢性病毒性肝炎的相关性研究作了综述.     

A contribution to the laboratory assay of typhoid vaccines

In the laboratory assay of typhoid vaccines many questions still remain open. The results of experiments are at times ambiguous, and there is no satisfactory agreement between them and the results of the various field trials. The authors of the present paper have attempted to clear up some of the causes of discrepancy. They have concluded that in evaluating the efficacy of various types of vaccine much depends on the vaccine and challenge doses used. By employing low or high doses of vaccine, it is possible to obtain different results in respect of the qualities of the vaccines compared. The time interval between vaccination and challenge also has a certain influence.     

The use of combination vaccinia vaccines and dual-gene vaccinia vaccines to enhance antigen-specific T-cell immunity via T-cell costimulation

Several recombinant vaccinia viruses are currently being evaluated to induce antigen-specific immunity to a variety of infectious disease agents and tumor associated antigens. T-cell costimulation is extremely important in enhancing T-cell responses, and recombinant vaccines have now been shown to be effective vectors to express a range of these molecules. Both combination vaccines (an admixture of a recombinant vaccinia virus expressing a specific target antigen and a recombinant vaccinia virus expressing a costimulatory molecule) and dual-gene vaccines expressing both transgenes on the same vector have been shown capable of effectively enhancing antigen-specific responses via T-cell costimulation. In this report, we compare for the first time the use of both types of approaches to enhance antigen-specific T-cell responses, and we demonstrate the importance of route of vaccine administration and vaccine dose in attaining optimal T-cell responses. These studies should have direct bearing on the design of vaccine clinical trials for infectious agents and/or tumor associated antigens, in which T-cell costimulatory molecules will be employed to enhance antigen-specific T-cell responses via the use of either combination or dual-gene vaccinia vaccines.     

Heterosubtypic immunity to influenza mediated by liposome adjuvanted H5N1 recombinant protein vaccines

A non-egg, non-culture based influenza vaccine that intervenes large influenza outbreaks and protects against heterosubtypic infections is needed. Candidates of such vaccine are likely to be conserved influenza virus proteins or their coding DNA. The vaccine must be conveniently produced at reasonable cost, safe, highly immunogenic and should be able to recall rapidly the immunological memory upon the antigenic re-exposure. In this study vaccines made of full length recombinant NP and M2 of the H5N1 influenza A virus were entrapped either alone or together into liposome (L) made of phosphatidylcholine and cholesterol. The vaccines (L-NP, L-M2 or L-NP + M2) and mocks (L or PBS) were safe without causing any adverse reaction in the intramuscularly injected mice. They were readily immunogenic at a single dose and a recalled response could be detected within one day post booster. Cytokine and antibody data indicated that the vaccines induced a Th1 bias immune response. NP containing vaccines stimulated a marked increase of cytotoxic lymphocytes, i.e., CD8⁺, intracellular IFNγ⁺ cells, while M2 containing vaccines elicited good antibody response which neutralized infectivity of heterologous influenza viruses. Although the three vaccines elicited different immunological defense factors; nevertheless, they similarly and readily abrogated lung histopathology mediated by viruses belonging to different H5N1 clade/subclade and heterosubtypes including swine H1N1 and human H1N1/2009 viruses. They protected the vaccinated mice against lethal challenges with mouse adapted avian H5N1 virus. The liposome adjuvanted vaccines which demonstrated high protective efficacy in mice warrant testing further in a non-rodent model as well as in humans.     

The induction of systemic and mucosal immunity to protein vaccines delivered through skin sites exposed to UVB

It has been reported that common mucosal immunity can be efficiently induced in mice following immunization through the skin with vaccine formulations containing either the active form of vitamin D, or chemical agents capable of locally enhancing cyclic adenosine monophosphate levels. Herein, we report that exposure of skin to ultraviolet radiation B (UVB) can be employed as a means to alter systemic humoral immune responses and to promote the induction of mucosal immunity to protein antigens delivered into UVB-exposed skin sites. Our data indicates that the skin, as a vaccination site, can be manipulated to allow efficient induction of common mucosal and systemic immune responses.     

Lactogenic immunity to transmissible gastroenteritis virus induced by a subunit immunogen

A subunit prepared from transmissible gastroenteritis (TGE) virus and used to immunize 24 gilts prior to farrowing induced production of specific antibody in the serum and milk. Challenge of pigs, two to seven days of age and suckling on the vaccinated gilts, with the Illinois strain of TGE virus resulted in morbidity of 28% and mortality of 4% as compared with 100 and 73%, respectively, for control piglets. Piglets nursing on a sow which had been immunized approximately 10 months previously were not protected, indicating that lactogenic immunity may be of short duration. Revaccination of this animal resulted in an anamnestic response.     

Characterization of humoral and cell-mediated immunity induced by mRNA vaccines expressing influenza hemagglutinin stem and nucleoprotein in mice and nonhuman primates

In response to immune pressure, influenza viruses evolve, producing drifted variants capable of escaping immune recognition. One strategy for inducing a broad-spectrum immune response capable of recognizing multiple antigenically diverse strains is to target conserved proteins or protein domains. To that end, we assessed the efficacy and immunogenicity of mRNA vaccines encoding either the conserved stem domain of a group 1 hemagglutinin (HA), a group 2 nucleoprotein (NP), or a combination of the two antigens in mice, as well as evaluated immunogenicity in naïve and influenza seropositive nonhuman primates (NHPs). HA stem-immunized animals developed a robust anti-stem antibody binding titer, and serum antibodies recognized antigenically distinct group 1 HA proteins. These antibodies showed little to no neutralizing activity in vitro but were active in an assay measuring induction of antibody-dependent cellular cytotoxicity. HA-directed cell-mediated immunity was weak following HA stem mRNA vaccination; however, robust CD4 and CD8 T cell responses were detected in both mice and NHPs after immunization with mRNA vaccines encoding NP. Both HA stem and NP mRNA vaccines partially protected mice from morbidity following lethal influenza virus challenge, and superior efficacy against two different H1N1 strains was observed when the antigens were combined. In vivo T cell depletion suggested that anti-NP cell-mediated immunity contributed to protection in the mouse model. Taken together, these data show that mRNA vaccines encoding conserved influenza antigens, like HA stem and NP in combination, induce broadly reactive humoral responses as well as cell-mediated immunity in mice and NHPs, providing protection against homologous and heterologous influenza infection in mice.     

Multigenotype HCV-NS3 recombinant vaccinia viruses as a model for evaluation of cross-genotype immunity induced by HCV vaccines in the mouse

Surrogate infections with HCV-recombinant vaccinia viruses (HCV-rVV) are a standard method to test the efficacy of hepatitis C virus (HCV) vaccine candidates in the mouse model. We established a panel of 16 HCV-rVV expressing the nonstructural protein 3 (NS3) of HCV genotypes 1a, 1b, 2, 3 and 4. Mice immunized with recombinant NS3 protein derived from HCV genotype 1b were challenged with the rVV. rVV-titers decreased up to 54-fold after subtype 1b challenge and up to 8.5-fold after subtype 1a challenge. No change was detected for genotype 2, 3, or 4. Our model is a convenient and reliable tool to analyze the induction of cross-genotype immunity by experimental vaccination of mice.     

The effect of pre-existing adenovirus-specific immunity on immune responses induced by recombinant adenovirus expressing glycoprotein D of bovine herpesvirus type 1

We investigated whether pre-existing adenovirus-specific immunity influenced the development of immunity to a foreign antigen expressed by recombinant adenovirus. Active adenovirus-specific immunity was induced in cotton rats by i.n. administration of wild type human adenovirus type 5 (HAd5) two weeks before immunisation with a HAd5 vector expressing the glycoprotein D (gD) of bovine herpesvirus type 1 (gD-dE3 recombinant adenovirus). Active adenovirus-specific immunity inhibited gD-specific immune responses, following either i.n. or gastrointestinal immunisation with gD-dE3. An inhibitory effect was present even if infection with HAd5 and immunisation with gD-dE3 were 13 weeks apart. Passive transfer of adenovirus specific antibodies to cotton rats one day before immunisation, however, did not significantly inhibit gD-specific immune responses induced by i.n. immunisation with gD-dE3. Repeated administration of an adenovirus vector, therefore, may have a limited ability to deliver antigen, while passive immunity to adenovirus may not interfere with the success of immunisation.     

Identification of carcinogens by measurement of cell-mediated immunity: I. Immunity induced by rat pancreas and colon carcinogens

Anti-tumor cell-mediated immunity was used as a measure of exposure to chemical carcinogens. The measurements involved determining the amount of cytotoxicity to cultured X-ray-induced rat small bowel adenocarcinoma cells exhibited by peripheral blood lymphoid-cells isolated from animals exposed to the carcinogens. Cytotoxicity was determined by the release of radioiodinated integral and peripheral proteins of the small bowel cancer cells. The studies indicate that exposures to 1,2-dimethylhydrazine (DMH) which induces colon adenocarcinomas and 7,12-dimethylbenz[a]anthracene (DMBA) implants which produce pancreatic adenocarcinomas, can be detected within a 6- to 72-day postexposure interval. Dose response studies of the DMH indicate exposures to as little as 100 μg (1.7 μmol) can be detected within this time interval although this amount is insufficient to produce detectable cancer. The present results further outline the specificity of the assay since it was found that tumors of mesodermally derived tissues (asbestos-induced peritoneal mesotheliomas) and nonspecific injury (70% surgical resection of ileum and jejunum) will not induce cytotoxicity. Our previous studies had shown ectodermal lesions also will not evoke responses; therefore, these findings suggest the measured anti-tumor immunity is specific to carcinogens which interact with entodermally derived tissue. Thus, the findings support our suggestion that measurement of anti-tumor immunity offers a rapid, sensitive, and specific technique for detecting carcinogenic substances.     

Functional activities of human antibody induced by the capsular polysaccharide or polysaccharide-conjugate vaccines against Haemophilus influenzae type b

To determine the in vitro and in vivo activity of human antibody induced by different Haemophilus influenzae type b (Hib) vaccines, pooled human antisera obtained from adults immunized with either polyribosyl ribitol phosphate (PRP) or PRP-conjugated with diphtheria toxoid (PRP-D) vaccines were evaluated for opsonic and protective activity against Hib. In vitro, opsonophagocytic studies revealed that PRP-D induced antisera were approximately 2.5-fold more effective than PRP-induced antisera in supporting neutrophil-mediated killing of an Hib strain. In vivo studies using the infant rat model or Hib disease revealed that the decay of PRP antibody was similar with PRP or PRP-D induced antisera in unchallenged rats. However, in infant rats challenged with Hib, the PRP induced antibody decayed more rapidly than the PRP-D induced antibody as shown by significantly shorter half-life of the former. The protective efficacy was significantly greater with the PRP-D induced antisera than with the PRP induced antisera. This finding was shown by the significantly lower incidence of bacteraemia and the 5-fold lower dose of antibody required for 50% protection against bacteraemia in rats receiving the PRP-D induced antisera. The findings suggest that antibody to PRP induced by PRP-D vaccine is more opsonic and protective against Hib, and also decays more slowly in infant rats challenged with Hib than does antibody induced by PRP vaccine. Further studies are needed to elucidate the reason(s) for this difference in functional activity and in half-life of PRP antibody induced by PRP or by PRP-D vaccines.