Characterization of Immune Responses Following Neonatal DNA Immunization: A Dissertation

Neonatal mice have immature immune systems with defects in several components of inflammatory, innate, and specific immune responses and develop a preferential T helper type 2 (Th2) response following immunization with many vaccine antigens. Although maternal antibody is the major form of protection from disease in early life when the neonatal immune system is still immature, the presence of maternal antibody also interferes with active immunization, placing infants at risk for severe bacterial and viral infection. Recent studies have suggested that immunizing with DNA plasmids encoding the vaccine antigen of interest is highly efficacious in a variety of adult animal models. However, similar extensive studies have not been conducted in infants. In this dissertation, we examine both the quantitative and qualitative differences between neonatal and adult humoral and cell-mediated immune responses in the presence or absence of maternal antibody. First, we wished to determine if one-day-old neonatal mice immunized with plasmid DNA expressing influenza A/PR/8/34 hemagglutinin (HA) by either intramuscular (i.m.) or gene gun (g.g.) inoculation were capable of generating humoral responses comparable to those in mice immunized as adults. We found that newborn mice developed stable, long-lived, protective anti-HA-specific IgG responses similar in titer to those of adult DNA-immunized mice. However, unlike the adult i.m. and g.g. DNA immunizations, which develop polarized IgG2a and IgG1 responses, respectively, mice immunized as neonates developed a variety of IgG1-, IgG2a-, and mixed IgG1/IgG2a responses regardless of the inoculation method. Boosting increased, but did not change these antibody profiles. We also found that, in contrast to the DNA immunizations, inoculations of newborn mice with an A/PR/8/34 viral protein subunit preparation failed to elicit an antibody response. Further, temporal studies revealed that both responsiveness to protein vaccination and development of polarized patterns of T help following DNA immunization appeared by 2 weeks of age. To determine if the disparity of polarized IgG responses between neonatal and adult DNA vaccinated mice was due to deficiencies in Th1 promoting cytokines, we addressed the ability of DNA encoding Th1 cytokines to bias the isotype of antibody raised by neonatal DNA immunization. We found that neonatal mice coimmunized with HA and either IL-12 or IFNγ-expressing DNAs developed IgG2a-biased immune responses, regardless of inoculation method, whereas these DNAs had no effect on IgG subtype patterns in adult DNA immunized mice. Consistent with the Th1-promoting effects of these cytokines, we also observed that codelivery of IL-12 or IFNγ DNAs raised T helper responses toward Th1 in mice immunized both as neonates or adults. Thus, codelivery of cytokine DNAs may be effective at tailoring immune responses depending on the required correlates of protection for a given pathogen. Finally, we addressed the effect of maternal antibody on the elicitation of humoral and cell-mediated immune responses. We tested the ability of i.m. and g.g. immunization with DNA expressing influenza HA and/or nucleoprotein (NP) to raise protective humoral and cellular responses in the presence and absence of maternal antibody. We found that neonatal mice born to influenza-immune mothers raised full antibody responses to NP but failed to generate antibody responses to HA. In contrast, the presence of maternal antibody did not affect the generation of long-lived CD4+ and CD8+ T cell responses to both HA and NP. Thus, maternal antibody did not affect cell-mediated responses, but rather it limited humoral responses, with the ability to limit the antibody response correlating with whether the DNA-expressed immunogen was localized in the plasma membrane or within the cell. We further observed that protection from influenza virus challenge was dependent on the presence of anti-HA IgG and was independent of the presence T cell responses. Taken together with other published studies, the data presented in this dissertation help better characterize the responses elicited by DNA vaccines at birth. This dissertation presents several novel observations including the temporal development of polarized IgG subtype responses, the ability of codelivered Th1 cytokine DNA to affect both antibody and T cell responses in the neonate, and the ability to generate humoral responses to intracellular, but not plasma membrane proteins, in the presence of maternal antibody. Furthermore, the data provides rationale for further development of DNA vaccines in the neonate

Vaccination with DNA plasmids expressing Gn coupled to C3d or alphavirus replicons expressing Gn protects mice against rift valley fever virus

Background: Rift Valley fever (RVF) is an arthropod-borne viral zoonosis. Rift Valley fever virus (RVFV) is an important biological threat with the potential to spread to new susceptible areas. In addition, it is a potential biowarfare agent. Methodology/Principal Findings: We developed two potential vaccines, DNA plasmids and alphavirus replicons, expressing the Gn glycoprotein of RVFV alone or fused to three copies of complement protein, C3d. Each vaccine was administered to mice in an all DNA, all replicon, or a DNA prime/replicon boost strategy and both the humoral and cellular responses were assessed. DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited high titer neutralizing antibodies that were similar to titers elicited by the live-attenuated MP12 virus. Mice vaccinated with an inactivated form of MP12 did elicit high titer antibodies, but these antibodies were unable to neutralize RVFV infection. However, only vaccine strategies incorporating alphavirus replicons elicited cellular responses to Gn. Both vaccines strategies completely prevented weight loss and morbidity and protected against lethal RVFV challenge. Passive transfer of antisera from vaccinated mice into naïve mice showed that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited antibodies that protected mice as well as sera from mice immunized with MP12. Conclusion/Significance: These results show that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn administered alone or in a DNA prime/replicon boost strategy are effective RVFV vaccines. These vaccine strategies provide safer alternatives to using live-attenuated RVFV vaccines for human use. © 2010 Bhardwaj et al

Therapeutic DNA vaccine induces broad T cell responses in the gut and sustained protection from viral rebound and AIDS in SIV-infected rhesus macaques.

Immunotherapies that induce durable immune control of chronic HIV infection may eliminate the need for life-long dependence on drugs. We investigated a DNA vaccine formulated with a novel genetic adjuvant that stimulates immune responses in the blood and gut for the ability to improve therapy in rhesus macaques chronically infected with SIV. Using the SIV-macaque model for AIDS, we show that epidermal co-delivery of plasmids expressing SIV Gag, RT, Nef and Env, and the mucosal adjuvant, heat-labile E. coli enterotoxin (LT), during antiretroviral therapy (ART) induced a substantial 2-4-log fold reduction in mean virus burden in both the gut and blood when compared to unvaccinated controls and provided durable protection from viral rebound and disease progression after the drug was discontinued. This effect was associated with significant increases in IFN-γ T cell responses in both the blood and gut and SIV-specific CD8+ T cells with dual TNF-α and cytolytic effector functions in the blood. Importantly, a broader specificity in the T cell response seen in the gut, but not the blood, significantly correlated with a reduction in virus production in mucosal tissues and a lower virus burden in plasma. We conclude that immunizing with vaccines that induce immune responses in mucosal gut tissue could reduce residual viral reservoirs during drug therapy and improve long-term treatment of HIV infection in humans

Four-gene-combination DNA vaccine protects mice against a lethal vaccinia virus challenge and elicits appropriate antibody responses in nonhuman primates

AbstractTwo major infectious forms of vaccinia virus (VACV) have been described: the intracellular mature virion (IMV), and the extracellular enveloped virion (EEV). Due to their stability in the environment, IMVs play a predominant role in host-to-host transmission, whereas EEVs play an important role in dissemination within the host. In a previous report, we demonstrated that mice vaccinated with VACV L1R (IMV immunogen) and A33R (EEV immunogen) were protected from a lethal poxvirus challenge. Vaccination with a combination of both genes conferred greater protection than either gene alone, suggesting that an immune response against both IMV and EEV is advantageous. Here, we report that in mice individually administered DNA vaccines with two different VACV immunogens, A27L (IMV immunogen) or B5R (EEV immunogen), failed to significantly protect; however, vaccination with a combination of both genes conferred a high level of protection. Mice were completely protected when vaccinated with a combination of four VACV genes (A27L + A33R + L1R + B5R). Rhesus macaques vaccinated with this four-gene-combination developed appropriate antibody responses to each protein. Antibody responses elicited by this vaccine cross-reacted with monkeypox virus orthologous proteins. These data indicate that a gene-based vaccine comprised of the VACV A27L + A33R + L1R + B5R genes may be a useful candidate to protect against other orthopoxviruses, including those that cause monkeypox and smallpox

Antibody and T cell responses induced in chickens immunized with avian influenza virus N1 and NP DNA vaccine with chicken IL-15 and IL-18.

e had examined the immunogenicity of a series of plasmid DNAs which include neuraminidase (NA) and nucleoprotein (NP) genes from avian influenza virus (AIV). The interleukin-15 (IL-15) and interleukin-18 (IL-18) as genetic adjuvants were used for immunization in combination with the N1 and NP AIV genes. In the first trial, 8 groups of chickens were established with 10 specific-pathogen-free (SPF) chickens per group while, in the second trial 7 SPF chickens per group were used. The overall N1 enzyme-linked immunosorbent assay (ELISA) titer in chickens immunized with the pDis/N1+pDis/IL-15 was higher compared to the chickens immunized with the pDis/N1 and this suggesting that chicken IL-15 could play a role in enhancing the humoral immune response. Besides that, the chickens that were immunized at 14-day-old (Trial 2) showed a higher N1 antibody titer compared to the chickens that were immunized at 1-day-old (Trial 1). Despite the delayed in NP antibody responses, the chickens co-administrated with IL-15 were able to induce earlier and higher antibody response compared to the pDis/NP and pDis/NP+pDis/IL-18 inoculated groups. The pDis/N1+pDis/IL-15 inoculated chickens also induced higher CD8+ T cells increase than the pDis/N1 group in both trials (P0.05) in inducing CD4+ and CD8+ T cells when co-administered with the pDis/IL-18 in both trials in comparison to the pDis/NP. Our data suggest that the pDis/N1+pDis/IL-15 combination has the potential to be used as a DNA vaccine against AIV in chickens

Vaccination with hemagglutinin or neuraminidase DNA protects BALB/c mice against influenza virus infection in presence of maternal antibody

<p>Abstract</p> <p>Background</p> <p>Maternal antibody is the major form of protection against disease in early life; however, its presence interferes with active immunization of offspring. In order to overcome the immunosuppression caused by maternal antibody, several immune strategies were explored in this paper using mouse model and influenza vaccines.</p> <p>Results</p> <p>The results showed that: i) when the offspring were immunized with the same vaccine as their mothers, whether inactivated or DNA vaccine, the presence of maternal antibody inhibited offspring immune response and the offspring could not be protected from a lethal influenza virus infection; ii) when the offspring, born to mothers immunized with inactivated vaccine, were immunized with NA DNA vaccine, the interference of maternal antibody were overcome and the offspring could survive a lethal virus challenge; iii) when the offspring were immunized with different DNA vaccine from that for their mothers, the interference of maternal antibody were also overcome. In addition, high-dose inactivated vaccine in maternal immunization caused partial inhibition in offspring when the offspring were immunized with HA DNA vaccine, while lower dose caused no significant immunosuppression.</p> <p>Conclusion</p> <p>To avoid the interference of maternal antibody in influenza vaccination of offspring, mothers and their offspring shall not be immunized with the same vaccine. If mothers are immunized with inactivated vaccine, NA DNA vaccine for the offspring shall be effective; and if mothers are immunized with HA (NA) DNA, NA (HA) DNA for the offspring shall be effective.</p

A single immunization with HA DNA vaccine by electroporation induces early protection against H5N1 avian influenza virus challenge in mice

<p>Abstract</p> <p>Background</p> <p>Developing vaccines for the prevention of human infection by H5N1 influenza viruses is an urgent task. DNA vaccines are a novel alternative to conventional vaccines and should contribute to the prophylaxis of emerging H5N1 virus. In this study, we assessed whether a single immunization with plasmid DNA expressing H5N1 hemagglutinin (HA) could provide early protection against lethal challenge in a mouse model.</p> <p>Methods</p> <p>Mice were immunized once with HA DNA at 3, 5, 7 days before a lethal challenge. The survival rate, virus titer in the lungs and change of body weight were assayed to evaluate the protective abilities of the vaccine. To test the humoral immune response induced by HA DNA, serum samples were collected through the eye canthus of mice on various days after immunization and examined for specific antibodies by ELISA and an HI assay. Splenocytes were isolated after the immunization to determine the antigen-specific T-cell response by the ELISPOT assay.</p> <p>Results</p> <p>Challenge experiments revealed that a single immunization of H5N1 virus HA DNA is effective in early protection against lethal homologous virus. Immunological analysis showed that an antigen-specific antibody and T-cell response could be elicited in mice shortly after the immunization. The protective abilities were correlated with the amount of injected DNA and the length of time after vaccination.</p> <p>Conclusion</p> <p>A single immunization of 100 μg H5 HA DNA vaccine combined with electroporation was able to provide early protection in mice against homologous virus infection.</p

Studies on antibody responses following neonatal immunization with influenza hemagglutinin DNA or protein

Neonatal mice have immature immune systems with defects in several components of inflammatory, innate, and specific immune responses and develop a preferential T helper type 2 response following immunization with many vaccine antigens. These studies were undertaken to determine whether 1-day-old neonatal mice immunized with plasmid DNA expressing influenza A/PR/8/34 hemagglutinin (H1) by either intramuscular (im) or gene gun (gg) inoculation were capable of generating humoral responses comparable to those in mice immunized as adults. The newborn mice developed stable, long-lived, protective anti-H1-specific IgG responses similar in titer to those of adult DNA-immunized mice. However, unlike the adult im and gg DNA immunizations, which develop polarized IgG2a and IgG1 responses, respectively, mice immunized as neonates developed a variety of IgG1, IgG2a, and mixed IgG1/IgG2a responses regardless of the inoculation method. Boosting increased but did not change these antibody profiles. In contrast to the DNA immunizations, inoculations of newborn mice with an A/PR/8/34 viral protein subunit preparation failed to elicit an antibody response. Temporal studies revealed that both responsiveness to protein vaccination and development of polarized patterns of T help following DNA immunization appeared by 2 weeks of age