Efficacy analysis of severe acute respiratory syndrome coronavirus 2 DNA vaccine and recombinant subunit vaccine inducing neutralizing antibodies in mice / 第二军医大学学报

Objective To investigate the efficacy of neutralizing antibodies induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) and spike (S) protein S1 subunit. Methods The SARS-CoV-2 RBD and mouse immunoglobulin G1 (IgG1) Fc fragment (mFc) fusion protein expression plasmid pVRCRBD- mFc was constructed and transfected into human embryonic kidney 293T cells. The RBD-mFc fusion protein in the cell supernatants was detected by Western blotting. The effect of RBD-mFc in cell supernatants and CHO recombinant S1-human IgG1 Fc (S1-hFc) fusion protein on SARS-CoV-2 infection was detected by microneutralization test. BALB/c mice were immunized with plasmid pVRC-RBD-mFc and S1-hFc fusion protein via intramuscular injection. Anti-S1 IgG antibodies in mouse sera were detected by enzyme-linked immunosorbent assay (ELISA), and the virus neutralization activity of mouse sera was detected by microneutralization test. Results The RBD-mFc fusion protein could be detected in the culture supernatants of 293T cells transfected with the plasmid pVRC-RBD-mFc, the concentrated supernatants and the S1- hFc fusion protein could inhibit SARS-CoV-2 infection on Vero E6 cells in a concentration-dependent manner. Anti-S1 IgG antibodies could be detected in the sera of mice immunized with plasmid pVRC-RBD-mFc and S1-hFc fusion protein, and the sera of both groups could neutralize SARS-CoV-2 infection. The serum antibody titers and virus neutralization activity of S1- hFc fusion protein immunized mice were significantly higher than those of plasmid pVRC-RBD-mFc immunized mice (both P<0.01). Conclusion Both SARS-CoV-2 RBD and S1 subunit may be used as effective vaccine antigens. Compared with DNA vaccine, recombinant subunit vaccine can induce neutralizing antibody more effectively..

Avances en el desarrollo de una vacuna efectiva contra Cryptosporidium parvum: una revisión de la literatura / Advances in the development of an effective vaccine against Cryptosporidium parvum: An review of the literature / Avanços no desenvolvimento de uma vacina eficaz contra Cryptosporidium parvum: uma revisão da literatura

Introducción. Cryptosporidium parvum es un parásito zoonótico altamente prevalente, asociado a enfermedad diarreica en población inmunocomprometida, niños y terneros menores de 30 días. Esta infección puede ocasionar deshidratación, alteración del estado de conciencia, retraso en el desarrollo global y, en algunos casos, la muerte del paciente. A pesar de la alta prevalencia de C. parvum, no existen medicamentos completamente efectivos ni una vacuna aprobada para prevenir dicha enfermedad. Objetivo. Realizar una revisión de la literatura sobre candidatos vacunales contra C. parvum. Método. Revisión documental mediante la búsqueda de la literatura de los últimos 20 años, disponible en las bases de datos PubMed central, WEB OF SCIENCE, Embase, REDALYC y LILACS. Resultados. Las vacunas atenuadas, recombinantes, basadas en ADN, expresadas en vectores bacterianos y sintéticas han mostrado resultados prometedores en la inducción de inmunogenicidad contra los antígenos de C. parvum, siendo el antígeno de superficie de 15 kilodaltons de Cryptosporidium parvum (cp15), el antígeno inductor de una mejor respuesta inmune celular y humoral en el modelo murino estudiado. Conclusión. Se espera que la incorporación de nuevas técnicas para la selección de antígenos promisorios y la ejecución de una gran cantidad de ensayos in vivo, favorezcan el desarrollo de una vacuna totalmente efectiva contra C. parvum. Aunque el camino para lograr este objetivo será largo y difícil, se convierte en la mejor alternativa para controlar una de las enfermedades de interés en salud pública, con mayor impacto en la población inmunocomprometida.

Introduction. Cryptosporidium parvum is a highly prevalent zoonotic parasite, associated with diarrheal disease in immunocompromised population, children and calves under 30 days. This infection is associa- ted to dehydration, delayed global development and, in some cases, the death of the patient. Despite the high prevalence of C. parvum, there are no fully effective medications and an approved vaccine to prevent such disease. Objective. To conduct a thorough review of the literature on vaccine candidates against C. parvum. Method Documentary review by searching the literature of the last 20 years, available in the central PubMed, WEB OF SCIENCE, Embase, REDALYC and LILACS databases. Results. Attenuated, recombinant, DNA-based, expressed in bacterial vectors and synthetic vaccines have shown promising results in inducing immunogenicity against C. parvum, being the Cryptospori- dium parvum 15 kiloDalton surface antigen (cp15), the antigen inducer of a better cellular and humoral immune response in the murine model studied. Conclusion. It is expected that the incorporation of new techniques for the selection of promising antigens and the execution of a large number of in vivo assays will favor the development of a fully effective vaccine against C. parvum. Although the way to achieve this goal will be long and difficult, it will become the best alternative to control one of the diseases with the greatest impact on the immu- nocompromised population.

Introdução. O Cryptosporidium parvum é um parasita zoonótico de alta prevalência associado à doença diarreica em populações imunocomprometidas, crianças e bezerros com menos de 30 dias. Essa infecção pode causar desidratação, alteração do estado de consciência, atraso no desenvolvi- mento global e, em alguns casos, a morte do paciente. Apesar da alta prevalência de C. parvum, não existem medicamentos totalmente eficazes e uma vacina aprovada para prevenir a doença. Objetivo. Realizar uma revisão literária dos candidatos à vacina contra C. parvum. Método. Revisão documental, mediante pesquisa da literatura dos últimos 20 anos, disponível nas bases de dados PubMed central, WEB OF SCIENCE, Embase, REDALYC e LILACS. Resultados. Vacinas atenuadas, recombinantes e baseadas em DNA, expressas em vetores bacteria- nos e sintéticos, mostraram resultados promissores na indução de imunogenicidade contra antígenos de C. parvum, sendo o antígeno de superfície de 15 kilodaltons de Cryptosporidium parvum (cp15) o antígeno indutor de uma melhor resposta imune celular e humoral no modelo murino estudado. Conclusão. Se espera que a incorporação de novas técnicas para a seleção de antígenos promissores e a execução de um grande número de ensaios in vivo favoreçam o desenvolvimento de uma vacina totalmente eficaz contra C. parvum. Embora o caminho para alcançar este objetivo seja longo e difícil, torna-se a melhor alternativa para controlar uma das doenças de interesse na saúde pública com maior impacto na população imunocomprometida.

Herpes Zoster DNA Vaccines with IL-7 and IL-33 Molecular Adjuvants Elicit Protective T Cell Immunity

Herpes zoster (HZ), or shingles, is caused by the reactivation of latent varicella-zoster virus (VZV) from the sensory ganglia when VZV-specific T-cell immunity is decreased because of aging or immunosuppression. In the present study, we developed HZ DNA vaccine candidates encoding VZV proteins and cytokine adjuvants, such as IL-7 and IL-33. We immunized C57BL/6 mice with DNA plasmids encoding VZV glycoprotein E (gE), immediate early (IE) 63, or IE62 proteins and found that robust VZV protein-specific T-cell responses were elicited by HZ DNA vaccination. Co-administration of DNA plasmids encoding IL-7 or IL-33 in HZ DNA vaccination significantly enhanced the magnitude of VZV protein-specific T-cell responses. Protective immunity elicited by HZ DNA vaccination was proven by challenge experiments with a surrogate virus, vaccinia virus expressing gE (VV-gE). A single dose of HZ DNA vaccine strongly boosted gE-specific T-cell responses in mice with a history of previous infection by VV-gE. Thus, HZ DNA vaccines with IL-7 and IL-33 adjuvants strongly elicit protective immunity.

Purification and characterization of monoclonal IgG antibodies recognizing Ebola virus glycoprotein

PURPOSE: The goal of this study was to purify and characterize Ebola virus glycoprotein (GP)-specific IgG antibodies from hybridoma clones. MATERIALS AND METHODS: For hybridoma production, mice were injected by intramuscular-electroporation with GP DNA vaccines, and boosted with GP vaccines. The spleen cells were used for producing GP-specific hybridoma. Enzyme-linked immunosorbent assay, Western blot assay, flow cytometry, and virus-neutralizing assay were used to test the ability of monoclonal IgG antibodies to recognize GP and neutralize Ebola virus. RESULTS: Twelve hybridomas, the cell supernatants of which displayed GP-binding activity by enzyme-linked immunosorbent assay and the presence of both IgG heavy and light chains by Western blot assay, were chosen as a possible IgG producer. Among these, five clones (C36-1, D11-3, D12-1, D34-2, and E140-2) were identified to secrete monoclonal IgG antibodies. When the monoclonal IgG antibodies from the 5 clones were tested for their antigen specificity, they recognized GP in an antigen-specific and IgG dose-dependent manner. They remained reactive to GP at the lowest tested concentrations (1.953–7.8 ng/mL). In particular, IgG antibodies from clones D11-3, D12-1, and E140-2 recognized the native forms of GP expressed on the cell surface. These antibodies were identified as IgG1, IgG2a, or IgG2b kappa types and appeared to recognize the native forms of GP, but not the denatured forms of GP, as determined by Western blot assay. Despite their GP-binding activity, none of the IgG antibodies neutralized Ebola virus infection in vitro, suggesting that these antibodies are unable to neutralize Ebola virus infection. CONCLUSION: This study shows that the purified IgG antibodies from 5 clones (C36-1, D11-3, D12-1, D34-2, and E140-2) possess GP-binding activity but not Ebola virus-neutralizing activity.

Preferential production of IgM-secreting hybridomas by immunization with DNA vaccines coding for Ebola virus glycoprotein: use of protein boosting for IgG-secreting hybridoma production

PURPOSE: The goal of this study was to investigate the utility of DNA vaccines encoding Ebola virus glycoprotein (GP) as a vaccine type for the production of GP-specific hybridomas and antibodies. MATERIALS AND METHODS: DNA vaccines were constructed to express Ebola virus GP. Mice were injected with GP DNA vaccines and their splenocytes were used for hybridoma production. Enzyme-linked immunosorbent assays (ELISAs), limiting dilution subcloning, antibody purification methods, and Western blot assays were used to select GP-specific hybridomas and purify monoclonal antibodies (MAbs) from the hybridoma cells. RESULTS: Twelve hybridomas, the cell supernatants of which displayed GP-binding activity, were selected by ELISA. When purified MAbs from 12 hybridomas were tested for their reactivity to GP, 11 MAbs, except for 1 MAb (from the A6-9 hybridoma) displaying an IgG2a type, were identified as IgM isotypes. Those 11 MAbs failed to recognize GP. However, the MAb from A6-9 recognized the mucin-like region of GP and remained reactive to the antigen at the lowest tested concentration (1.95 ng/mL). This result suggests that IgM-secreting hybridomas are predominantly generated by DNA vaccination. However, boosting with GP resulted in greater production of IgG-secreting hybridomas than GP DNA vaccination alone. CONCLUSION: DNA vaccination may preferentially generate IgM-secreting hybridomas, but boosting with the protein antigen can reverse this propensity. Thus, this protein boosting approach may have implications for the production of IgG-specific hybridomas in the context of the DNA vaccination platform. In addition, the purified monoclonal IgG antibodies may be useful as therapeutic antibodies for controlling Ebola virus infection.

Improvement of DNA vaccination by adjuvants and sophisticated delivery devices: vaccine-platforms for the battle against infectious diseases

Advantages of DNA vaccination against infectious diseases over more classical immunization methods include the possibilities for rapid manufacture, fast adaptation to newly emerging pathogens and high stability at ambient temperatures. In addition, upon DNA immunization the antigen is produced by the cells of the vaccinated individual, which leads to activation of both cellular and humoral immune responses due to antigen presentation via MHC I and MHC II molecules. However, so far DNA vaccines have shown most efficient immunogenicity mainly in small rodent models, whereas in larger animals including humans there is still the need to improve effectiveness. This is mostly due to inefficient delivery of the DNA plasmid into cells and nuclei. Here, we discuss technologies used to overcome this problem, including physical means such as in vivo electroporation and co-administration of adjuvants. Several of these methods have already entered clinical testing in humans.

Cellular immunity survey against urinary tract infection using pVAX/fimH cassette with mammalian and wild type codon usage as a DNA vaccine

PURPOSE: FimH (the adhesion fragment of type 1 fimbriae) is implicated in uropathogenic Escherichia coli (UPEC) attachment to epithelial cells through interaction with mannose. Recently, some studies have found that UPEC can thrive intracellularly causing recurrent urinary tract infection (UTI). Almost all vaccines have been designed to induce antibodies against UPEC. Yet, the humoral immune response is not potent enough to overcome neither the primary UTI nor recurrent infections. However, DNA vaccines offer the possibility of inducing cell mediated immune responses and may be a promising preventive tool. MATERIALS AND METHODS: In this study, we employed two different open reading frames within mammalian (mam) and wild type (wt) codons of fimH gene. Optimized fragments were cloned in pVAX-1. Expression of the protein in COS-7 was confirmed by western blot analysis after assessing pVAX/fimH(mam) and pVAX/fimH(wt). The constructs were injected to BALB/c mice at plantar surface of feet followed by electroporation. RESULTS: The mice immunized with both constructs following booster injection with recombinant FimH showed increased interferon-gamma and interleukin-12 responses significantly higher than non-immunized ones (p<0.05). The immunized mice were challenged with UPEC and then the number of bacteria recovered from the immunized mice was compared with the non-immunized ones. Decreased colony count in immunized mice along with cytokine responses confirmed the promising immune response by the DNA vaccines developed in this study. CONCLUSION: In conclusion, DNA vaccines of UPEC proteins may confer some levels of protection which can be improved by multiple constructs or boosters.

DNA immunization of Mycobacterium tuberculosis resuscitation-promoting factor B elicits polyfunctional CD8+ T cell responses

PURPOSE: T cell-mediated immune responses, and particularly activation of polyfunctional T cells that simultaneously produce multiple cytokines, are necessary for the control of Mycobacterium tuberculosis. In the present study, we examined if DNA immunization of Mycobacterium tuberculosis resuscitation-promoting factor B (RpfB) elicits polyfunctional T cell responses in mice. MATERIALS AND METHODS: C57BL/6 mice were immunized intramuscularly three times, at 3-week intervals, with RpfB-expressing plasmid DNA. For comparison, protein immunization was performed with recombinant RpfB in control mice. After immunization, RpfB-specific T cell responses were assessed by interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot assay and intracellular cytokine staining (ICS), and T cell polyfunctionality was assessed from the ICS data. RESULTS: RpfB DNA immunization induced not only humoral immune responses, but also CD8+ and CD4+ T cell responses. Immunodominant T-cell epitopes were identified within RpfB by assays with overlapping peptides. RpfB DNA immunization elicited a polyfunctional CD8+ T cell response that was dominated by a functional phenotype of IFN-gamma+/TNF-alpha+/IL-2-/CD107a+. CONCLUSION: RpfB DNA immunization elicits polyfunctional CD8+ T cell responses, suggesting that RpfB DNA immunization might induce protective immunity against tuberculosis.

Immunogenicity and efficacy of a plasmid DNA rabies vaccine incorporating Myd88 as a genetic adjuvant

PURPOSE: Myeloid differentiation factor 88 (Myd88), a ubiquitous Toll-like receptor adaptor molecule, has been reported to play important roles in B cell responses to infections and vaccination. The present study evaluated the effects of genetic adjuvanting with Myd88 on the immune responses to a plasmid DNA rabies vaccine. MATERIALS AND METHODS: Plasmids encoding rabies glycoprotein alone (pIRES-Rgp) or a fragment of Myd88 gene in addition (pIRES-Rgp-Myd) were constructed and administered intramuscularly or intrademally in Swiss albino mice (on days 0, 7, and 21). Rabies virus neutralizing antibody (RVNA) titres were estimated in the mice sera on days 14 and 28 by rapid fluorescent focus inhibition test. The protective efficacy of the constructs was evaluated by an intracerebral challenge with challenge virus standard virus on day 35. RESULTS: Co-expression of Myd88 increased RVNA responses to pIRES-Rgp by 3- and 2-folds, following intramuscular and intradermal immunization, respectively. pIRES-Rgp protected 80% of the mice following intramuscular and intradermal immunizations, while pIRES-Rgp-Myd afforded 100% protection following similar administrations. CONCLUSION: Genetic adjuvanting with Myd88 enhanced the RVNA responses and protective efficacy of a plasmid DNA rabies vaccine. This strategy might be useful for rabies vaccination of canines in the field, and needs further evaluation.

Microneedle patches for vaccine delivery

In today's medical industry, the range of vaccines that exist for administration in humans represents an eclectic variety of forms and immunologic mechanisms. Namely, these are the live attenuated viruses, inactivated viruses, subunit proteins, and virus-like particles for treating virus-caused diseases, as well as the bacterial-based polysaccharide, protein, and conjugated vaccines. Currently, a new approach to vaccination is being investigated with the concept of DNA vaccines. As an alternative delivery route to enhance the vaccination efficacy, microneedles have been devised to target the rich network of immunologic antigen-presenting cells in the dermis and epidermis layers under the skin. Numerous studies have outlined the parameters of microneedle delivery of a wide range of vaccines, revealing comparable or higher immunogenicity to conventional intramuscular routes, overall level of stability, and dose-sparing advantages. Furthermore, recent mechanism studies have begun to successfully elucidate the biological mechanisms behind microneedle vaccination. This paper describes the current status of microneedle vaccine research.

DNA vaccines targeting human papillomavirus-associated diseases: progresses in animal and clinical studies

Human papillomavirus (HPV) infection is a major cause of cervical cancer and its precancerous diseases. Cervical cancer is the second deadliest cancer killer among women worldwide. Moreover, HPV is also known to be a causative agent of oral, pharyngeal, anal and genital cancer. Recent application of HPV structural protein (L1)-targeted prophylactic vaccines (Gardasil(R) and Cervarix(R)) is expected to reduce the incidence of HPV infection and cervical cancer, and possibly other HPV-associated cancers. However, the benefit of the prophylactic vaccines for treating HPV-infected patients is unlikely, underscoring the importance of developing therapeutic vaccines against HPV infection. In this regard, numerous types of therapeutic vaccine approaches targeting the HPV regulatory proteins, E6 and E7, have been tested for their efficacy in animals and clinically. In this communication, we review HPV vaccine types, in particular DNA vaccines, their designs and delivery by electroporation and their immunologic and antitumor efficacy in animals and humans, along with the basics of HPV and its pathogenesis.

Avaliação do potencial protetor da vacina gênica pvaxapa- dmt-me no modelo experimental de tuberculose / Protective potential evaluation of genetic vaccines pvaxapa- dmt-me in the experimental model of tuberculosis

A tuberculose (TB) é responsável por dois milhões de mortes a cada ano, apesar daampla utilização da vacina BCG (Bacilo Calmette-Guérin). Embora essa vacina nãoproteja contra a TB pulmonar no adulto, protege contra as formas graves da TB nainfância. Para muitos autores, a imunização heteróloga (prime-boost) talvezseja uma das estratégias mais importantes e realistas para o controle da TB,principalmente nos países endêmicos. Muitos estudos têm demonstrado que aimunização com BCG seguido de reforço com vacina gênica (genes de M.tuberculosis) induz melhor proteção contra TB do que somente a vacina BCG. Alémdisso, a estratégia prime-boost utilizando microesferas biodegradáveis pareceser uma imunização promissora para estimular a resposta imune de longa duração.A glicoproteína APA foi identificada como importante antígeno imunodominante,induzindo níveis significativos de proteção contra M. tuberculosis. Assim, esteestudo teve como objetivo avaliar a eficácia protetora da imunização heteróloga emmodelo experimental de TB. Para isso, camundongos BALB/c foram imunizados porvia subcutânea com BCG seguido de um reforço por via intramuscular com aformulação vacinal contendo apa e DMT co-encapsuladosem microesferas (BCG/APA). Essa estratégia prime-boost foi eficiente na indução daresposta imune protetora à infecção por M. tuberculosis, caracterizada pela reduçãosignificativa do número de bacilos no pulmão destes animais na fase mais tardia dainfecção (70 dias após desafio). Além de ser significativamente mais imunogênciaque a vacina BCG, permitiu uma melhor preservação do parênquima pulmonar, comredução do número e tamanho dos granulomas, limitando a extensão da injúriatecidual induzida pela infecção e restringindo a inflamação no tecido alveolar. Apotencialização do efeito protetor da vacina BCG induzida pelo reforço com a vacinapVAXapa+DMT-Me sugere que a estratégia utilizada pode ser importante para aprevenção da TB...

Tuberculosis (TB) is responsible for two millions of deaths each year despite thewidespread use of BCG vaccine (Bacillus Calmette-Guérin). Although this vaccinedoes not protect against adult pulmonary TB, it is protective against severe forms ofchildhood TB. For many authors, heterologous prime-boost regimen is perhaps themost realistic strategy for future TB control, especially in endemic countries. Manystudies have demonstrated that BCG-prime DNA-(M. tuberculosis genes)-boosterinduce greater protection against TB than BCG alone. APA glycoprotein has beenidentified as major immunodominant antigen and induces significant levels ofprotection against M. tuberculosis. Moreover, prime-boost strategy by usingbiodegradable microspheres seems to be a promising immunization to stimulate longlasting immune response. Thus, this study had as aim to investigate the immuneprotection against M. tuberculosis challenge in mice based on BCG priming and DNAvaccine boosting. For that, BALB/c mice were inoculated subcutaneously with aheterologous BCG priming-DNA boosting immunization using pVAXapa and TDM coencapsulatedin microspheres (BCG/APA), intramuscularly. This prime-boost strategywas effective in inducing protective immune response against M. tuberculosisinfection, characterized by significant lower numbers of bacilli in the lungs of theseanimals, in the late phase of infection (70 days after infection). In addition to beingsignificantly more immunogenic than BCG, it allowed better preservation of the lungparenchyma and reduction in the number and size of granulomas, limiting the extentof lung injury induced by infection and inflammation in the alveolar tissue. Theimprovement of the protective effect of BCG vaccine mediated by pVAXapa boostersuggests that our strategy can be important in the treatment for prevention of TB...

A novel DNA vaccine constructed by heat shock protein 70 and melanoma antigen-encoding gene 3 against tumorigenesis.

Melanoma antigen-encoding gene 3 (MAGE-3) is an ideal candidate for a tumor vaccine although its potency need to be increased. Heat shock proteins (HSPs) represents a potential approach for increasing the potency of DNA vaccines. In the present study, a fusion DNA vaccine composed of Mycobacterium tuberculosis HSP70 and MAGE-3 was constructed and used to immunize C57BL/6 mice against B16 or B16-MAGE-3 tumor cells. The results show that the HSP70-MAGE-3 fusion DNA vaccine enhanced the frequency of MAGE-3-specific cytotoxic T-cells as compared to the MAGE-3 DNA vaccine or the HSP70/MAGE-3 cocktail DNA vaccine (P<0.05). In conclusion, the results indicate that the HSP70-MAGE-3 fusion DNA vaccine can strongly activate MAGE-3 specific cellular immunological reactions and thus significantly inhibit the growth of B16-MAGE-3 tumors, improving the survival of tumor-bearing mice, and the HSP70-MAGE-3 fusion DNA vaccine has a significant therapeutic effect on the tumors that express MAGE-3 antigens.

DNA vaccines encoding hemagglutinin of novel H1N1 influenza A virus inducing neutralization antibody in mice / 中华微生物学和免疫学杂志

Objective To study the characteristics of neutralization antibody in mice induced by DNA vaccines of hemagglutinin(HA) of novel H1N1 influenza A virus(2009H1N1).Methods HA encoding plasmids of 2009H1N1 or 1918H1N1(2009HA or 1918HA)were constructed.25 μg or 200 μg dosage of 2009HA plasmids were used to immunize the mice,the total antibody of anti-20O9HA or cross-reactive antibody were assayed by ELISA using 2009HA or 1918HA protein as capture antigen,and the neutralizing antibody were assayed by two kinds of virus pseudo - particles(pp) of 2009H1N1 and 1918H1N1 .Results During of 4 to 16 weeks after boost immunization,in two groups of mice immunized with 25 μg or 200 μg dosage 2009HA plasmids,both total antibody of anti-2009HA and neutralizing antibody to 2009H1Nlpp reached the similar level(P ＞0.05),and there were cross-reactive antibody to 1918HA protein in two groups of mice serum,with similar titers of cross-neutralizing activity to 1918H1N1 pp(P ＞0.05),Conclusion A low dosage DNA vaccine encoding HA of 2009 H1 N1 virus is able to induce persistent and high level of neutralizing antibody,and may be potential valuable vaccine against the new emerging influenza virus.

Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP) fused antigens: a potential tool to develop DNA vaccines against flaviviruses

Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the developent of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP). The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.

A vacinação é a estratégia mais prática e o melhor custo-benefício para prevenir a maioria das infecções dos flavivirus, para os quais existe vacina disponível. Entretanto, as vacinas baseadas em vírus atenuados podem potencialmente promover efeitos colaterais e, mais raramente, reações fatais. Diante deste cenário, o desenvolvimento de estratégias alternativas de vacinação, como vacinas baseadas em DNA codificando seqüências específicas dos flavivirus, está sendo considerado. Antí-genos citoplasmáticos endógenos, caracteristicamente codificados por vacinas de DNA plasmidial, são majoritariamente apresentados ao sistema imune através de moléculas do Complexo Maior de Histocompatibilidade de classe I - MHC I. A via de apresentação MHC I é mais associada à resposta celular citotóxica e, frequentemente, não elicita uma resposta humoral satisfatória. Uma das principais estratégias para direcionar antígenos codificados pelas vacinas de DNA para o compartimento MHC II é expressar estes antígenos dentro da Proteína de Associação à Membrana Lisossomal (LAMP). A proteína do envelope dos flavivirus é reconhecidamente a principal proteína de superfície viral e o principal alvo para anticorpos neutralizantes. Diferentes grupos têm demonstrado que a co-expressão das proteínas de membrana e do envelope dos flavivirus em células de mamíferos, fusionada com a porção carboxi-terminal de LAMP, é capaz de induzir níveis satisfatórios de anticorpos neutralizantes. Neste trabalho revisamos a estratégia de co-expressão da proteína do envelope dos flavivírus, como quimeras de LAMP, com o objetivo de desenvolver vacinas de DNA contra a febre do Oeste do Nilo, dengue e febre amarela.

The study on the immunogenicity of Streptococcus pneumoniae pneumolysin DNA vaccine in Rhesus macaques / 中华微生物学和免疫学杂志

Objective To study the immunogenicity of Streptococcus pneumoniae pneumolysin DNA vaccine in Rhesus macaques. Methods The deletion of the gene sequence encoding for the 11 amino acids at the carboxyl terminal of pneumolysin (PN) from its wild type gene (pn) by PCR resulted in a mu-tant pneumolysin gene (pnd). The wild type pn gene encoding PN and the mutant gene (pnd) encoding PND were cloned into pVAX1 vector respectively and then tested as Ppn and Ppnd DNA vaccines. The PN and PND proteins were purified from recombinant E. coli. Rhesus macaques were immunized by intramuscu-larly (i.m.) injection of Ppn or Ppnd DNA vaccine with electroporation (EP). Results Because of the deletion of the gene sequence encoding for the eleven amino acids at the carboxyl terminal of the PN from pn gene, the recombinant PND antigen lost its hemolytic activity while its antigenicity was remained. The spe-cific humoral immunity against pneumolysin was induced by injecting monkey with 500 μg DNA followed by EP. Boosting the Ppn or Ppnd DNA/EP primed animals with corresponding recombinant protein, PN or PND, evoked strong immune response at about 4 fold increase in the antibody titer. Conclusion Specific antibody responses were induced in the monkeys by DNA vaccination and electroporation. The immunogenic-ity of the DNA vaccines were significantly enhanced when PN or PND protein boost was applied 10 d after third DNA vaccination.

Forecasting system on spatial coverage of cumulative phenotypic frequency of HLA class Ⅰ for designing HLA-based vaccines in China / 免疫学杂志

Objective To build the forecasting system on spatial coverage of cumulative phenotypic frequency (CPF) of HLA- Ⅰ for designing HLA-based vaccines (epitopes or DNA vaccines) and evaluating their effects in China. Methods The spatial database of HLA- Ⅰgenes of Chinese (across the land of China) was set up, and then the spatial forecasting system of CPF was designed using Kriging technique.Results Using this spatial forecasting system, the vaccine designer could predict the spatial coverage of CPF for any combination of the alleles confined to a single class Ⅰ locus, either HLA-A or HLA-B, as well as for any combination of alleles at these two loci of each given population at any geographical location across the land of China. Conclusions This system is applicable in the following rields: ①To provide for the identification of alleles that represent a desired percentage of populations across the land of China and that can be targeted for vaccine composition. ② To predict the theoretical responder status of vaccines whose HLA restricted epitopes have already been known in given population at any geographical location across the land of China. (③) To identify how many individuals will be non-responders to a HLA-based vaccine across the land of China.

Enhancing protective immunity effects of the vaccine against Schistosoma japonicum infection through priming with cocktail DNA vaccines and boosting with cocktail protein vaccines / 中国血吸虫病防治杂志

Objective To enhance the protective immunity effects against Schistosoma japonicum infection by priming with cocktail DNA vaccines and boosting with cocktail protein vaccines in infected BALB/c mice.Methods Plasmids and proteins for immunization were prepared and diluted in no bacterial saline solution to final concentration of 1.5 mg/ml,and mixed with pcDNA3.1-SjC23,pcDNA3.1-SjCTPI,pcDNA3.1-(CDR3)6 plasmid DNAs by equal volume to form the cocktail DNA vaccine,and also mixed with recombinant proteins SjC23-HD,SjCTPI,and NP30 by equal volume to form the cocktail protein vaccine.Seventy female BALB/c mice of 4-5 weeks old were randomly divided into 5 groups(A,B,C,D,E).In Group A(control group),each mouse was immunized with 100 ?l saline solution by intramuscular(i.m.);in Group B(pcDNA3.1 control group),each mouse was immunized(i.m.)with 100 ?l pcDNA3.1 for three times at week 0,3,6;in Group C(pcDNA3.1 and cocktail protein group),each mouse was immunized(i.m.)with 100 ?l pcDNA3.1 for three times at week 0,3,6 and immunized with 100 ?l mixed protein vaccines plus 100 ?l FCA by subcutaneous at week 9;in Group D(cocktail DNA vaccines group),each mouse was immunized(i.m.)with 100 ?l mixed DNA vaccines for three times at week 0,3,6;in Group E(cocktail DNA vaccines plus cocktail proteins),each mouse was immunized(i.m.)with 100 ?l mixed DNA vaccines for three times at week 0,3,6 and immunized with 100 ?l mixed protein vaccines plus 100 ?l FCA by subcutaneous at week 9.Four weeks after the last DNA immunization or two weeks after protein boosting,all the mice were challenged with(40?1)cercariae of Schistosoma japonicum by abdominal skin penetration at the same time.Forty-two days post-challenge,the mice were sacrificed and perfused,and the numbers of recovered worms and eggs in liver were counted.The blood was collected from the tail veins of all the mice two days before the first immunization and challenge,respectively,the serum was prepared for detection of IgG,IgG1 and IgG2a.Two days before the challenge,the spleen cells of two mice from each group were cultured and stimulated with ConA and soluble egg antigen(SEA),and the supernatant was collected for detection of IL-2,IL-4 and IFN-?.Results The worm reduction rates in Group C,D and E were 17.70%,32.88% and 45.35%,respectively,compared with the control group.The worm reduction rates in Group D and E were significantly higher than that in Group C(P

Immunological effects of different adjuvants on HCV-DNA vaccine / 中国免疫学杂志

Objective:The immunological effects of HCV-DNA vaccine with different adjuvants were detected by ELISPOT in mice.Methods:Female BALB/c mice were primed with naked HCV-DNA, HCV-DNA encapsulated by liposome DDAB/EPC or DC-Chol/DOPE, HCV-DNA mixed with Montanide ISA 720 or aluminum hydroxide, respectively, and boosted twice accordingly in a four-week interval. Cytokine production by splenocytes was assessed by ELISPOT.Results:In most cases, splenocytes from mice vaccinated with DDAB/EPC liposome produced more IFN-?. These splenocytes also have significant higher IL-2 production compared with the other groups. In expansion with NS5b, splenocytes from alum group have significance in IL-4 production compared with other groups. The profile of cytokine production revealed that the INF-? overwhelmed IL-4 in naked DNA, DDAB/EPC, and DC-Chol/DOPE groups while IL-4 surmounted IFN-? in alum and Montanide groups.Conclusion:Encapsulation with liposome DDAB/EPC has the strongest adjuvant effect in inducing Th1 dominated immunity. Alum and Montanide can convert the Th1 nature of DNA vaccine to Th2-biased immunity.