Cellular Immune Responses In Mice Research Articles

Mechanisms of Naturally Acquired Immunity to Streptococcus pneumoniae.

In this review we give an update on the mechanisms of naturally acquired immunity against Streptococcus pneumoniae, one of the major human bacterial pathogens that is a common cause of pneumonia, septicaemia, and meningitis. A clear understanding of the natural mechanisms of immunity to S. pneumoniae is necessary to help define why the very young and elderly are at high risk of disease, and for devising new prevention strategies. Recent data has shown that nasopharynx colonization by S. pneumoniae induces antibody responses to protein and capsular antigens in both mice and humans, and also induces Th17 CD4+ cellular immune responses in mice and increases pre-existing responses in humans. These responses are protective, demonstrating that colonization is an immunizing event. We discuss the data from animal models and humans on the relative importance of naturally acquired antibody and Th17 cells on immunity to S. pneumoniae at three different anatomical sites of infection, the nasopharynx (the site of natural asymptomatic carriage), the lung (site of pneumonia), and the blood (site of sepsis). Mouse data suggest that CD4+ Th17 cells prevent both primary and secondary nasopharyngeal carriage with no role for antibody induced by previous colonization. In contrast, antibody is necessary for prevention of sepsis but CD4+ cellular responses are not. Protection against pneumonia requires a combination of both antibody and Th17 cells, in both cases targeting protein rather than capsular antigen. Proof of which immune component prevents human infection is less easily available, but two recent papers demonstrate that human IgG targeting S. pneumoniae protein antigens is highly protective against septicaemia. The role of CD4+ responses to prior nasopharyngeal colonization for protective immunity in humans is unclear. The evidence that there is significant naturally-acquired immunity to S. pneumoniae independent of anti-capsular polysaccharide has clinical implications for the detection of subjects at risk of S. pneumoniae infections, and the data showing the importance of protein antigens as targets for antibody and Th17 mediated immunity should aid the development of new vaccine strategies.

Vaccination with recombinant adenoviruses expressing Toxoplasma gondii MIC3, ROP9, and SAG2 provide protective immunity against acute toxoplasmosis in mice

Toxoplasmosis is a worldwide zoonosis caused by the protozoan parasite Toxoplasma gondii, an obligate intracellular parasite. Currently, no viable vaccine or effective drug strategies exist to prevent and control toxoplasmosis. T. gondii microneme protein 3 (MIC3), rhoptry protein 9 (ROP9), and surface antigen 2 (SAG2) are related to active invasion of the parasite. Hence, we constructed recombinant adenoviruses expressing TgMIC3, TgSAG2, or TgROP9 and evaluated the recombinant adenoviruses as potential vaccines against acute T. gondii infection in BALB/c mice. Mice immunized with the recombinant adenoviruses were measured the antibody levels, percentages of lymphocytes and actived T lymphocytes, cytokine productions, and the survival rates and time to evaluate the protective efficacy. Results showed that immunization with the bivalent or trivalent recombiant adenoviruses could strongly stimulate humoral and cellular immune responses in mice, resulting in effective immune protections against lethal challenge with the tachyzoites of T. gondii RH. These results indicated that the divalent and trivalent adenoviruses, especially Ad-ROP9-MIC3-EGFP, may be promising vaccine candidates against acute T. gondii infection.

Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) enhances the immune responses against Helicobacter pylori in BALB/c mice

Objective To evaluate the adjuvant activities of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) in enhancing humoral and cellular responses against Helicobacter pylori (H.pylori). Methods BALB/c mice were immunized with the protein antigens including UreA, UreB and NapA of H. pylori in combination with cGAMP as the adjuvant on 0 d and 14 d by subcutaneous administration. Then, the serum-specific antibody responses were evaluated by ELISA. Flow cytometry (FCM) and enzyme-linked immunospot assay (ELISpot) were used to detect the cellular immune responses occurred in spleen and mesenteric lymph nodes (MLN). Results Subcutaneous administration of protein antigens of H. pylori together with cGAMP induced strong humoral and cellular immune responses in BALB/c mice. The levels of serum-specific IgG antibodies induced by adding cGAMP as the adjuvant were significantly higher than those by immunizing with antigens alone. The levels of splenic IFN-γ-producing lymphocytes in response to H. pylori antigens and cGAMP immunization were significantly higher than those in the corresponding groups without using cGAMP. Conclusion By using cGAMP as an adjuvant, H. pylori antigens could elicit significantly stronger humoral and cellular immune responses in mice than those induced by the antigens only. As a stable small molecular compound with strong adjuvant activity, cGAMP has the potential to be used for the development of H. pylori vaccine. Key words: cGAMP; Adjuvant; Helicobacter pylori; Vaccine

Immune responses elicited by the recombinant Erp, HspR, LppX, MmaA4, and OmpA proteins from Mycobacterium tuberculosis in mice.

Immunogenic potency of the recombinant Erp, HspR, LppX, MmaA4, and OmpA proteins from Mycobacterium tuberculosis (MTB), formulated with Montanide ISA 720 VG adjuvant, was evaluated in BALB/c mice for the first time in this study. The five vaccine formulations, adjuvant, and BCG vaccine were subcutaneously injected into mice, and the sera were collected at days 0, 15, 30, 41, and 66. The humoral and cellular immune responses against vaccine formulations were determined by measuring serum IgG and serum interferon-gamma (IFN-γ) and interleukin-12 (IL-12) levels, respectively. All formulations significantly increased IgG levels post-vaccination. The highest increase in IFN-γ level was provided by MmaA4 formulation. The Erp, HspR, and LppX formulations were as effective as BCG in enhancement of IFN-γ level. The most efficient vaccine boosting the IL-12 level was HspR formulation, especially at day 66. Erp formulation also increased the IL-12 level more than BCG at days 15 and 30. The IL-12 level boosted by MmaA4 formulation was found to be similar to that by BCG. OmpA formulation was inefficient in enhancement of cellular immune responses. This study showed that MmaA4, HspR, and Erp proteins from MTB are successful in eliciting both humoral and cellular immune responses in mice.

Escherichia coli-Derived Outer Membrane Vesicles Deliver Galactose-1-Phosphate Uridyltransferase and Yield Partial Protection against Actinobacillus pleuropneumoniae in Mice.

In our previous studies, we have identified several in vivo-induced antigens and evaluated their potential as subunit vaccine candidates in a murine model, in which the recombinant protein GalT showed the most potent immunogenicity and immunoprotective efficacy against Actinobacillus pleuropneumoniae. To exploit a more efficient way of delivering GalT proteins, in this study, we employed the widely studied E. coli outer membrane vesicles (OMVs) as a platform to deliver GalT protein and performed the vaccine trial using the recombinant GalT-OMVs in the murine model. Results revealed that GalT-OMVs could elicit a highly-specific, IgG antibody titer that was comparable with the adjuvant GalT group. Significantly higher lymphocyte proliferation and cytokines secretion levels were observed in the GalT-OMVs group. 87.5% and 50% of mice were protected from a lethal dose challenge using A. pleuropneumoniae in active or passive immunization, respectively. Histopathologic and immunohistochemical analyses showed remarkably reduced pathological changes and infiltration of neutrophils in the lungs of mice immunized with GalT-OMVs after the challenge. Taken together, these findings confirm that OMVs can be used as a platform to deliver GalT protein and enhance its immunogenicity to induce both humoral and cellular immune responses in mice.

Oral immunization with recombinant Lactococcus lactis NZ9000 expressing human papillomavirus type 16 E7 antigen and evaluation of its immune effects in female C57BL/6 mice.

The ORFs of both native and codon-optimized E7 genes were successfully fused to SPusp45 signal peptide and expressed by a nisin-controlled gene expression system in the NZ9000 strains of Lactococcus lactis. Recombinant strains were confirmed by Western blot analysis. To measure immune responses against the E7 antigen, specific-pathogen-free C57BL/6 mice were inoculated with L lactis harboring pNZ8123-rE7 by oral gavage. Then, specific antibodies and cytokines were measured by enzyme-linked immunosorbent assay and enzyme-linked immunospot assay, respectively. Oral administration of L lactis strains expressing rE7 elicited the highest levels of E7-specific antibody and greatest numbers of E7-specific CD4+ T helper and CD8+ T cell precursors. Our outcomes indicated that the HPV-16 E7 specific IL-2- and IFN-γ-secreting T cells in antigen-stimulated splenocytes and intestinal mucosal lymphocytes were significantly higher than the control groups. Our data also demonstrated that mice vaccinated with recombinant L lactis were able to generate potent protective effects against challenge with the E7-expressing tumor cell line (TC-1). Moreover, L lactis containing pNZ8123-HPV16-optiE7 showed strong therapeutic antitumor effects against established tumors in vivo. These findings demonstrate that recombinant L lactis induce both humoral and cellular immune responses in mice and are therefore recommended for therapeutic treatments in humans after oral administration.

Microneedles Improve the Immunogenicity of DNA Vaccines

DNA vaccines can elicit both humoral and cellular immune responses in mice. However, their poor immunogenicity is a major obstacle toward clinical applications. Improving the efficiency of delivery of DNA vaccines has become a key issue. Vaccination via microneedles penetrating the epidermis can dramatically enhance the stimulation of immune responses. This study showed that by using microneedles to deliver DNA vaccines, gene expression and corresponding immune responses were greatly improved compared to conventional needle injection. The quantitative analysis of gene expressions was made at 6, 24, 72, and 144 h after the DNA delivery. DNA expression levels increased in a time-dependent manner and were substantially greater than with syringe injection after 6 h and 24 h. This elevated expression was followed by markedly enhanced immune responses, with 6-10 times higher levels of antibody and T-cell responses.

Influenza virus-like particles composed of conserved influenza proteins and GPI-anchored CCL28/GM-CSF fusion proteins enhance protective immunity against homologous and heterologous viruses

Influenza viruses cause significant morbidity and mortality and pose a substantial threat to public health. Vaccination represents the principle means of preventing influenza virus infection. Current vaccine approaches are hindered by the need to routinely reformulate vaccine compositions in an effort to account for the progressive antigenic changes that occur as influenza viruses circulate in the human population. In this study, we evaluated chimeric virus-like particle (cVLP) vaccines containing conserved elements of influenza proteins (HL5M2e (HA stem gene with 5M2e gene inserted) and NP), with or without glycosylphosphatidylinositol-anchored CCL28 (GPI-CCL28) and/or GM-CSF (GPI-GM-CSF) fusion proteins as molecular adjuvants. cVLPs elicited strong humoral and cellular immune responses against homologous and heterologous viruses, and improved survival following lethal challenge with both homologous and heterologous viruses. Inclusion of GPI-anchored adjuvants in cVLP vaccines augmented the generation of influenza-specific humoral and cellular immune responses in mice in comparison to the non-adjuvanted cVLP vaccines. VLPs containing GPI-anchored adjuvants reduced morbidity and improved survival to lethal challenge with homologous and heterologous influenza viruses. This work suggests that VLP vaccines incorporating conserved influenza virus proteins and GPI-anchored molecular adjuvants may serve as a platform for a broadly protective “universal” influenza vaccine.

Immune response elicited by graphene oxide-based nanovaccine

Objective To study the antigen-specific immune response induced by the graphene oxide (GO) in mice. Methods OVA-loaded GO nano-immunocomplexes (GO-OVA) were prepared by co-incubation of nano GO with model antigen ovalbumin (OVA). Nano GO was characterized by atomic force microscopy and laser particle size analyzer. The cytotoxicity of GO to mouse bone marrow dendritic cells (BMDCs) was detected by cell counting kit (CCK-8). The GO-OVA uptake of BMDCs were observed by fluorescent staining. C57BL/6 mice were divided into OVA group, aluminum adjuvant OVA (Al-OVA) group and GO-OVA group (6 mice in each group) by body weight for in vivo immunization. The levels of OVA-specific antibody IgG (total IgG, IgG1, and IgG2a) in serum of mice were detected by enzyme-linked immunosorbent assay (ELISA). The T lymphocyte subsets in spleen and inguinal lymph nodes of mice were detected by flow cytometry. Results The average particle size of the prepared nano GO was (294.34±4.68) nm, and the polydispersity coefficient was 0.208. Nano GO has less toxicity to mouse BMDCs. The results of in vitro experiments indicated that GO-OVA nanovaccine can be efficiently internalized by mouse BMDCs. The antigen-specific IgG antibodies induced by the GO-OVA was similar to that of aluminum adjuvant and the difference was not statistically significant (P>0.05), and the Th1-type response was predominant. The proportions of CD4+ and CD8+ T lymphocytes in the spleen and inguinal lymph nodes in GO-OVA group were significantly higher than those in OVA and Al-OVA groups, and the differences were statistically significant (all P<0.05). Conclusions GO-OVA nano-immunocomplexes can induce both humoral and cellular immune responses in mice, which provides basis for the development of novel vaccine vectors and adjuvants. Key words: Graphene oxide; Nanovaccine; Humoral immune response; Cellular immune response

Immunization with Pseudomonas aeruginosa outer membrane vesicles stimulates protective immunity in mice

Pseudomonas aeruginosa is an opportunistic pathogen responsible for a wide range of severe nosocomial and community acquired infections, these infections are major health problems for cystic fibrosis patients and immune-compromised individuals. The emergence of multidrug-resistant isolates highlights the need to develop alternative strategies for treatment of P. aeruginosa infections. Outer membrane vesicles (OMVs) are spherical nanometer-sized proteolipids that are secreted from numerous of pathogenic Gram-negative bacteria, and a number of studies have confirmed the protective efficacy for use of OMVs as candidate vaccines. In this study, OMVs from P. aeruginosa (PA_OMVs) were isolated, formulated with aluminum phosphate adjuvant and used as a vaccine in a mouse model of acute lung infection. The results confirmed that active immunization with PA_OMVs was able to reduce bacterial colonization, cytokine secretion and tissue damage in the lung tissue, thus protecting mice from lethal challenge of P. aeruginosa. Cytokines assay validated that immunization with PA_OMVs was efficient to induce a mixed cellular immune response in mice. Further, high level of specific antibodies was detected in mice immunized with PA_OMVs, and results from opsonophagocytic killing assay and passive immunization suggested that humoral immune response may be critical for PA_OMVs mediated protection. These findings demonstrated that PA_OMVs may be served as a novel candidate vaccine for the prevention of P. aeruginosa infection.

Alphavirus Replicon DNA Vectors Expressing Ebola GP and VP40 Antigens Induce Humoral and Cellular Immune Responses in Mice.

Ebola virus (EBOV) causes severe hemorrhagic fevers in humans, and no approved therapeutics or vaccine is currently available. Glycoprotein (GP) is the major protective antigen of EBOV, and can generate virus-like particles (VLPs) by co-expression with matrix protein (VP40). In this study, we constructed a recombinant Alphavirus Semliki Forest virus (SFV) replicon vector DREP to express EBOV GP and matrix viral protein (VP40). EBOV VLPs were successfully generated and achieved budding from 293 cells after co-transfection with DREP-based GP and VP40 vectors (DREP-GP+DREP-VP40). Vaccination of BALB/c mice with DREP-GP, DREP-VP40, or DREP-GP+DREP-VP40 vectors, followed by immediate electroporation resulted in a mixed IgG subclass production, which recognized EBOV GP and/or VP40 proteins. This vaccination regimen also led to the generation of both Th1 and Th2 cellular immune responses in mice. Notably, vaccination with DREP-GP and DREP-VP40, which produces both GP and VP40 antigens, induced a significantly higher level of anti-GP IgG2a antibody and increased IFN-γ secreting CD8+ T-cell responses relative to vaccination with DREP-GP or DREP-VP40 vector alone. Our study indicates that co-expression of GP and VP40 antigens based on the SFV replicon vector generates EBOV VLPs in vitro, and vaccination with recombinant DREP vectors containing GP and VP40 antigens induces Ebola antigen-specific humoral and cellular immune responses in mice. This novel approach provides a simple and efficient vaccine platform for Ebola disease prevention.

Induction of Humoral and Cellular Immune Responses in Mice by Multiepitope Vaccines Composing of Both T and B Lymphocyte Epitopes of MAGE-A3 which are Recombined into HBcAg.

Melanoma-associated antigen-A3 (MAGE-A3) is a tumor specific antigen and a potential candidate for cancer immunotherapy. We had screened three immunodominant multiepitopes of MAGE-A3, and identified these multiepitope peptides had significantly higher reactivity to serum samples from gastric cancer patients. However, the immune responses of three multiepitope peptides carried by HBcAg in mice have not been investigated. The main objective of this study was to analyze the humoral and cellular immune responses in mice induced by these three multiepitope vaccines of MAGE-A3. Three multiepitopes of MAGE-A3 (MAGE-A3(EPI-1, or -2, or -3)) were respectively inserted at HBcAg major immunodominant region (HBcAg(MIR)) of the pET21a(+)/HBcAg(MIR) recombinant plasmid. These recombinant chimeras were identified by PCR, and transfected respectively into E. Coli Ressotta strain. The expression products of rHBcAg(MIR)/MAGE-A3(EPI-1, or -2, or -3) were purified respectively by Ni2+ chelated affinity column, and then confirmed by SDS-PAGE and Western-blot analysis.Purified three rHBcAg(MIR)/MAGE-A3 multiepitopes were administrated respectively into BALB/c (H-2Kd) mice by intradermal injection. The production of rHBcAg(MIR)/MAGE-A3(EPI-1, or -2, or -3) specific IgG in serum from immunized mice were measured by ELISA. Spleen cells from all immunized mice were harvested after one week of last immunization for lymphocyte proliferation assay and cytotoxic T-lymphocyte assay. PCR and Sequencing analysis showed the presence of the required gene fragment in pET21a(+)/ HBcAg(MIR)/MAGE-A3(EPI-1, or -2, or -3) recombinant plasmid. Purified rHBcAg(MIR)/MAGE-A3(EPI-1, or -2, or -3) could be probed specifically by McAb of 6×his-tag. ELISA analysis indicated that serum from immunized mice with rHBcAg(MIR)/MAGE-A3(EPI-1, -2, or -3) proteins could be discerned specifically by complete MAGE-A3 protein, and high level of antibodies in immune serum were obtained, and all antibody titers could reach above 1:1600. The splenocytes from groups of rHBcAg(MIR)/MAGE-A3(EPI-1,-2, or -3), stimulated respectively with corresponding peptides showed the higher proliferative responses comparing with control groups of HBcAg(MIR) or PBS (p<0.05, respectively). Splenocytes from mice immunized with rHBcAg(MIR)/MAGE-A3 (EPI-1, or -2, or -3) could killed target cells effectively, and there were significant difference of CTL activities compared with control groups of HBcAg(MIR), or PBS (p<0.05, respectively) at any ratio of effector : target. Our results indicated MIR in HBcAg presenting platform could present MAGE-A3 multiepitopes efficiently and induced significant humoral or cellular immunity. The immune strategy based on multiepitopeimmunization could have potential for preventing or controlling MAGE-A3 associated malignant disease.

Humoral and cellular immune response in mice induced by the classical swine fever virus E2 protein fused to the porcine CD154 antigen

The development of subunit vaccines against classical swine fever is a desirable goal, because it allows discrimination between vaccinated and infected animals. In this study, humoral and cellular immune response elicited in inbred BALB/c mice by immunization with a recombinant classical swine fever virus (CSFV) E2 protein fused to porcine CD154 antigen (E2CD154) was assessed. This model was used as a predictor of immune response in swine. Mice were immunized with E2CD154 emulsified in Montanide ISA50V2 or dissolved in saline on days 1 and 21. Another group received E2His antigen, without CD154, in the same adjuvant. Montanide ISA50V2 or saline served as negative controls for each experimental group. Animals immunized with 12.5 and 2.5 μg/dose of E2CD154 developed the highest titers (>1:2000) of CSFV neutralizing antibodies. Moreover, CSFV specific splenocyte gamma-interferon production, measured after seven and twenty-eight days of immunization, was significantly higher in mice immunized with 12.5 μg of E2CD154. As a conclusion, E2CD154 emulsified in Montanide ISA50 V2 was able to induce a potent humoral and an early cellular immune response in inbred BALB/c mice. Therefore, this immunogen might be an appropriate candidate to elicit immune response in swine, control CSF disease and to eliminate CSFV in swine.

Vaccination with Combination DNA and Virus-Like Particles Enhances Humoral and Cellular Immune Responses upon Boost with Recombinant Modified Vaccinia Virus Ankara Expressing Human Immunodeficiency Virus Envelope Proteins.

Heterologous prime boost with DNA and recombinant modified vaccinia virus Ankara (rMVA) vaccines is considered as a promising vaccination approach against human immunodeficiency virus (HIV-1). To further enhance the efficacy of DNA-rMVA vaccination, we investigated humoral and cellular immune responses in mice after three sequential immunizations with DNA, a combination of DNA and virus-like particles (VLP), and rMVA expressing HIV-1 89.6 gp120 envelope proteins (Env). DNA prime and boost with a combination of VLP and DNA vaccines followed by an rMVA boost induced over a 100-fold increase in Env-specific IgG antibody titers compared to three sequential immunizations with DNA and rMVA. Cellular immune responses were induced by VLP-DNA and rMVA vaccinations at high levels in CD8 T cells, CD4 T cells, and peripheral blood mononuclear cells secreting interferon (IFN)-γ, and spleen cells producing interleukin (IL)-2, 4, 5 cytokines. This study suggests that a DNA and VLP combination vaccine with MVA is a promising strategy in enhancing the efficacy of DNA-rMVA vaccination against HIV-1.

Humoral and cellular immune response of mice challenged with Yersinia pestis antigenic preparations

ObjectivesThe plague, which is an infectious disease caused by Yersinia pestis, still threatens many populations in several countries. The worldwide increase in human plague cases and the potential use of the bacteria as a biological weapon reinforce the need to study the immunity that is induced by potential vaccine candidates. To determine the immunogenicity of antigenic preparations based on the F1 protein and the total extract from Y. pestis, we assessed the role of these antigens in inducing an immune response. MethodsThe immunogenicity of antigenic preparations based on the Y. pestis (YP) total extract and the Y. pestis fraction 1 capsular antigen protein (F1) was determined in Swiss-Webster mice immunized with 40μg or 20μg for each preparation. Immunophenotyping was performed by flow cytometry. ResultsAnimals immunized with the YP total extract did not elicit detectable anti-F1 antibodies (Ab) in the hemaglutination/inhibition (HA/HI) test. Animals immunized with 40μg or 20μg of the F1 protein produced anti-F1 Abs, with titres ranging from 1/16 to 1/8132. The average of CD3+–CD4+ and CD3+–CD8+ T cells did not differ significantly between the groups. Neither YP total extract nor F1 protein induced a significant expression of IFN-γ and IL-10 in CD4+ T lymphocytes. In addition, F1 failed to induce IFN-γ expression in CD8+ T cells, unlike the YP total extract. ConclusionThe results showed that F1 protein is not an immunogenic T cell antigen, although the YP total extract (40μg dose) favoured CD8+ T cell-mediated cellular immunity.

Heterologous prime-boost immunization with live SPY1 and DnaJ protein of Streptococcus pneumoniae induces strong Th1 and Th17 cellular immune responses in mice

Streptococcus pneumoniae is a leading cause of infectious diseases in children under 5-year-old. Vaccine has been used as an indispensable strategy to prevent S. pneumoniae infection for more than 30 years. Our previous studies confirmed that mucosal immunization with live attenuated strain SPY1 can protect mice against nasopharyngeal colonization of S. pneumoniae and lethal pneumococcal infection, and the protective effects are comparable with those induced by commercially available 23-valent polysaccharide vaccine. However, live attenuated vaccine SPY1 needs four inoculations to get satisfactory protective effect, which may increase the risk of virulence recovery. It is reported that heterologous primeboost approach is more effective than homologous primeboost approach. In the present study, to decrease the doses of live SPY1 and improve the safety of SPY1 vaccine, we immunized mice with SPY1 and DnaJ protein alternately. Our results showed that heterologous prime-boost immunization with SPY1 and DnaJ protein could significantly reduce the colonization of S. pneumoniae in the respiratory tract of mice, and induce stronger Th1 and Th17 cellular immune responses than SPY1 alone. These results indicate heterologous prime-boost immunization method not only elicits better protective effect than SPY1 alone, but also reduces the doses of live SPY1 and decreases the risk of SPY1 vaccine. This work is the first time to study the protective efficiency with two different forms of S. pneumoniae candidate vaccine, and provides a new strategy for the development of S. pneumoniae vaccine.

Plant-produced Zika virus envelope protein elicits neutralizing immune responses that correlate with protective immunity against Zika virus in mice.

SummaryThe global Zika virus (ZIKV) outbreak and its link to foetal and newborn microcephaly and severe neurological complications in adults call for the urgent development of ZIKV vaccines. In response, we developed a subunit vaccine based on the ZIKV envelope (E) protein and investigated its immunogenicity in mice. Transient expression of ZIKV E (zE) resulted in its rapid accumulation in leaves of Nicotiana benthamiana plants. Biochemical analysis revealed that plant‐produced ZIKV E (PzE) exhibited specific binding to a panel of monoclonal antibodies that recognize various zE conformational epitopes. Furthermore, PzE can be purified to >90% homogeneity with a one‐step Ni2+ affinity chromatography process. PzE are found to be highly immunogenic, as two doses of PzE elicited both potent zE‐specific antibody and cellular immune responses in mice. The delivery of PzE with alum induced a mixed Th1/Th2 immune response, as the antigen‐specific IgG isotypes were a mixture of high levels of IgG1/IgG2c and splenocyte cultures from immunized mice secreted significant levels of IFN‐gamma, IL‐4 and IL‐6. Most importantly, the titres of zE‐specific and neutralizing antibodies exceeded the threshold that correlates with protective immunity against multiple strains of ZIKV. Thus, our results demonstrated the feasibility of plant‐produced ZIKV protein antigen as effective, safe and affordable vaccines against ZIKV.

Formulation of chitosan with the polyepitope HIV-1 protein candidate vaccine efficiently boosts cellular immune responses in mice.

Human immunodeficiency virus-1 (HIV-1) continues to be a major global public health issue and priority. Despite the variety of antiretroviral therapies, it seems that an effective vaccine against HIV-1 is still very necessary. An ideal HIV-1 vaccine should be able to elicit both humoral and cellular immunities. In this respect, polyepitope vaccines, incorporated from several conserved regions of HIV-1 proteins, have received much attention recently. Herein, the immunogenicity of the HIV-1 polyepitope protein-based candidate vaccines was evaluated in BALB/c mice. Following the plasmid (pET23a-HIV-1-tat/pol/gag/env) preparation and transformation, the recombinant protein expression was optimized in Escherichia coli BL21 (DE3) host cells. After the HIV-1-top4 protein purification, chitosan and alum adjuvants were added to the vaccines formulations to reinforce the immunogenicity of the candidate vaccines. Mice were subcutaneously immunized three times at 2-week intervals with the candidate vaccines and the elicitation of both humoral and cellular immune responses were investigated. Taken together, the results showed that chitosan adjuvanted candidate vaccine conferred a stronger immunogenicity and elicited higher cellular responses than other candidate vaccines (P<0.05). Thereby, it seems that co-utilizing of potent adjuvants with the HIV-1 polyepitope protein vaccines can help to open new avenues for strategies for HIV/AIDS vaccine design.

Influence of Individual Mutations in Genes Coding Internal Proteins of the Influenza A Virus on Formation of Humoral and Cellular Immune Response in Mice

This study addresses a current problem of vaccinal prevention—the development of approaches to increase the immunogenicity of influenza vaccines—and it is directed at studying the effect of mutations responsible for the degree of attenuation of influenza viruses on the formation of an immune response. We conducted the analysis of the humoral and cellular immune response in mice with the introduction of strains of the influenza A virus containing single and combined point mutations alike typical for the attenuation donor A/Leningrad/134/17/57 (H2N2), which is used at present for the preparation of domestic live influenza vaccine. In the study, 13 mutant strains were compared to the attenuation donor (containing all these mutations) and its “wild” predecessor (without mutations). It has been shown that the presence in internal genes of the “wild” virus of single mutations that are typical for the attenuation donor, as well as their combinations, can affect not only the quantitative indices of the humoral immune response, but also the rate of accumulation of virus-specific serum and secretory antibodies. A group of viruses with mutations in the M1 gene was isolated based on the ability to stimulate T cell response. A promising approach to development of ways to increase the immunogenic properties of live influenza vaccines will be a further search for a balanced combination of mutations in M1 and NS2 genes that enhance the stimulation of most of the immuneresponse factors studied, with mutations in genes of polymerase complex that impart attenuating properties to strains.

The tetravalent formulation of domain III-capsid proteins recalls memory B- and T-cell responses induced in monkeys by an experimental dengue virus infection.

Tetra DIIIC is a vaccine candidate against dengue virus (DENV) composed by four chimeric proteins that fuse the domain III of the envelope protein of each virus to the corresponding capsid protein. Containing B- and T-cell epitopes, these proteins form aggregates after the incubation with an immunostimulatory oligodeoxynucleotide, and their tetravalent formulation induces neutralizing antibodies and cellular immune response in mice and monkeys. Also, Tetra DIIIC protects mice after challenge with each DENV, and the monovalent formulation obtained from DENV-2 protects monkeys upon homologous viral challenge. However, in the last years, new evidences have arisen regarding domain III of DENV envelope protein as irrelevant target for neutralizing antibodies in humans. Nevertheless, vaccination with domain III induces a neutralizing antibody response that confers protection against re-infection. In addition, it has been demonstrated that the induction of a cellular immune response is essential to protect during the infection. This response can also avoid severe manifestations of dengue disease, associated to the antibody-dependent enhancement of the infection. In this study, we observed that Tetra DIIIC was able to boost the antiviral and neutralizing antibody responses previously generated in monkeys during an experimental DENV infection, demonstrating that domain III is targeted by B cells during the viral infection. Additionally, Tetra DIIIC successfully boosted the cellular immune response generated by the viruses, probably against T-cells epitopes in the capsid proteins. These results highlight the functionality of Tetra DIIIC as a vaccine candidate against DENV.