Measles virus DNA vaccination: antibody isotype is determined by the method of immunization and by the nature of both the antigen and the coimmunized antigen

Plasmids encoding the measles virus hemagglutinin (HA) and nucleoprotein (NP) proteins inoculated into the skin of BALB/c mice by the gene gun method induced both humoral and cytotoxic lymphocyte class I-restricted immune responses. Although intramuscular immunization induces the immunoglobulin G2a (IgG2a) antibody isotype for both antigens, with gene gun immunization, the NP still generated mainly IgG2a and the major isotype induced by the HA was IgG1. Interestingly, gene gun coimmunization of HA and NP plasmids resulted in a dominant IgG1 HA response and the switching of antibodies generated against the NP to the IgG1 isotype.     

Cytotoxic T cell responses to DNA vaccination: dependence on antigen presentation via class II MHC

This study was designed to test whether cytotoxic T cell (CTL) responses to DNA vaccination are dependent upon MHC class II-restricted priming of CD4+ T cells. Because DNA vaccination may directly transfect dendritic cells, and dendritic cells may be capable of directly stimulating CD8+ T cell responses, such priming might be unnecessary. To test this hypothesis, C57BL/6 mice were immunized intramuscularly or intradermally with DNA encoding either whole OVA, a class I (Kb)-restricted peptide epitope of OVA (amino acids 257-264, SIINFEKL), or this class I-restricted epitope plus the adjacent class II (I-Ab)-restricted epitope of OVA (amino acids 265-280, TEWTSSNVMEERKIKV). Very low to negligible CTL responses were observed in mice vaccinated with the SIINFEKL construct, whereas mice vaccinated with the SIINFEKLTEWTSSNVMEERKIKV or with the complete OVA construct made equally robust CTL responses. These responses were sensitive to blocking by anti-CD8 mAb and were shown to be SIINFEKL-specific by using SIINFEKL peptide-pulsed EL-4 cells as targets. To ensure that the generation of these CTL responses was indeed dependent upon CD4+ T cell help, mice were depleted of either CD4+ or CD8+ cells before immunization. Depletion of CD4+ cells completely abrogated the CTL response to OVA DNA, as did depletion of CD8+ cells. Thus, we conclude that the CTL response to both intramuscular and intradermal DNA vaccination is highly dependent upon the generation of CD4+ T cell help via a class II MHC-dependent pathway. These results will be relevant for the construction of minimal-epitope vaccines for DNA immunization.     

Protective cytotoxic T lymphocyte responses against paramyxoviruses induced by epitope-based DNA vaccines: involvement of IFN-gamma

Plasmid DNA vectors have been constructed with minigenes encoding a single cytotoxic T lymphocyte (CTL) epitope from either the M2 protein of respiratory syncytial virus (RSV) or from the nucleoprotein of measles virus (MV) with or without a signal sequence (also called secretory or leader sequence). Following intradermal immunization, plasmids in which the CTL epitopes were expressed in-frame with the signal sequence were more effective at inducing peptide- and virus-specific CTL responses than plasmids expressing CTL epitopes without the signal sequence. This immunization resulted in protection against MV-induced encephalitis and a significant reduction in viral load following RSV challenge. The reduction of viral load following RSV challenge was abrogated by prior injection with anti-IFN-gamma antibodies. These results highlight the ability of epitope-based DNA immunization to induce protective immune responses to well-defined epitopes and indicate the potential of this approach for the development of vaccines against infectious diseases.     

The effect of epitope variation on the profile of cytotoxic T lymphocyte responses to the HIV envelope glycoprotein

To address the relationship between viral and host factors during HIV infection, we analyzed the effect of viral mutations on T cell responses in seropositive, asymptomatic HLA-A2+ individuals using four envelope (env)-specific peptides with the HLA-A*0201 binding motif. We showed that the natural sequence variation was frequent within epitopes located in the C-terminal region of the env glycoprotein and was largely responsible for a lower env-specific cytotoxic T lymphocyte (CTL) activity in the peptide-stimulated cultures. The highest CTL responses in vitro were induced with conserved epitopes D1 and 4.3 that mapped to the N-terminal region of the env glycoprotein. These peptides exhibited high binding affinity for HLA-A*0201 molecules and stimulated CD8+ T cells of relatively limited TCR Vbeta chain repertoire. Decreased CTL activities to the D1 epitope were observed in the absence of any detectable viral mutation, and were associated with lower proliferative responses and expression of the CD28 antigen. Results of this study demonstrate that the degree of sequence variation within a stimulatory epitope of the viral quasispecies, as well as proliferative potential of the effector cells, are among the factors underlying decreased CTL activity in HIV-infected patients. These experiments also provide evidence that the D1 peptide might be useful for the development of vaccines and immune-based therapy.     

Antigen expressed on tumor cells fails to elicit an immune response, even in the presence of increased numbers of tumor-specific cytotoxic T lymphocyte precursors

We have used T-cell receptor (TCR) transgenic mice to analyze the interaction of tumors with the immune system. We show that the tumor cell line Lewis lung-lymphocytic choriomeningitis virus (LL-LCMV), genetically manipulated to express an H-2 Db-restricted epitope of the lymphocytic choriomeningitis virus glycoprotein (LCMV33-41), can grow progressively in TCR transgenic mice, where approximately 50% of CD8+ T cells are specific for LCMV33-41. TCR transgenic T cells were not deleted in tumor-bearing mice, and their surface phenotype and cytokine secretion patterns remained typical of naive T cells. Also, TCR transgenic T cells from tumor-bearing mice had undiminished capacity to proliferate to antigen in vitro. Tumor-protective immune responses could be elicited in TCR transgenic mice by immunization with LCMV33-41 peptide-loaded dendritic cells. Tumor resistance correlated with the switch of TCR transgenic T cells from a CD44low to a CD44high phenotype and increased capacity to produce IFNgamma in vitro. Results similar to those obtained in TCR transgenic mice were also obtained using an adoptive transfer system, where small numbers of TCR transgenic T cells were injected into normal C57BL/6 hosts. These data indicate that even large tumors may not induce specific immunization, tolerance, or anergy to tumor antigens, and that high numbers of tumor-specific CTL precursors are not sufficient to provide tumor resistance.     

Influence of maternal antibodies on vaccine responses: inhibition of antibody but not T cell responses allows successful early prime-boost strategies in mice

The transfer of maternal antibodies to the offspring and their inhibitory effects on active infant immunization is an important factor hampering the use of certain vaccines, such as measles or respiratory syncytial virus vaccine, in early infancy. The resulting delay in protection by conventional or novel vaccines may have significant public health consequences. To define immunization approaches which may circumvent this phenomenon, experiments were set up to further elucidate its immunological bases. The influence of maternal antibodies on antibody and T cell responses to measles hemagglutinin (MV-HA) were analyzed following MV-HA immunization of pups born to immune or control BALB/c mothers using four different antigen delivery systems: live or inactivated conventional measles vaccine, a live recombinant canarypox vector and a DNA vaccine. High levels (> 5 log10) of maternal anti-HA antibodies totally inhibited antibody responses to each of the vaccine constructs, whereas normal antibody responses were elicited in presence of lower titers of maternal antibodies. However, even high titers of maternal antibodies affected neither the induction of vaccine-specific Th1/Th2 responses, as assessed by proliferation and levels of IFN-gamma and IL-5 production, nor CTL responses in infant mice. On the basis of these unaltered T cell responses, very early priming and boosting (at 1 and 3 weeks of age, respectively) with live measles vaccine allowed to circumvent maternal antibody inhibition of antibody responses in pups of immune mothers. This was confirmed in another immunization model (tetanus toxoid). It suggests that effective vaccine responses may be obtained earlier in presence of maternal antibodies through the use of appropriate immunization strategies using conventional or novel vaccines for early priming.     

Protective efficacy of a dengue 2 DNA vaccine in mice and the effect of CpG immuno-stimulatory motifs on antibody responses

A recently described DNA vaccine for dengue (DEN) type 2 was shown to elicit high levels of neutralizing antibodies in mice. The vaccine candidate consists of the PreM and 92% of the envelope genes of DEN 2 New Guinea C strain. We further evaluated this DNA vaccine candidate by examining the effect of immuno-stimulatory CpG DNA motifs on antibody response and by studying the protective efficacy of the vaccine. The results showed that CpG motifs present in pUC 19 significantly improved the antibody response to a suboptimal dose of 3.1 micrograms of the DEN DNA vaccine. In a lethal mouse intracerebral challenge model, the vaccine provided a significant level of protection. Sixty percent of the mice immunized with the DEN DNA vaccine plus pUC 19 survived the challenge compared to only 10% in the control group that received vector plus pUC. These studies illustrate that nucleic acid immunization is a viable approach to developing a DEN vaccine and that immuno-stimulatory CpG DNA motifs can be used to lower the minimum dose required to produce an antibody response.     

Influence of the specific T cell response on seroconversion after measles vaccination in autologous bone marrow transplant patients

Six patients who were seronegative to measles after autologous bone marrow transplantation (ABMT) were vaccinated with a live attenuated measles vaccine. The specific T helper cell response was studied by measuring lymphocyte proliferation induced by measles antigen and B cell response by measles specific IgG by ELISA. Blood samples were drawn before, at 1-3 months, and at 1 year after vaccination. It was found that a pre-existing T cell response correlated with an impaired B cell response 1 year after vaccination (r = 0.83, P = 0.04), whereas no correlation was found between IgG titers before vaccination and IgG titer increase, or T cell response after vaccination. Furthermore, there was a transient negative correlation between the T cell response at 1-3 months after vaccination and the T cell response before vaccination (r = -0.90, P = 0.04) that became positive at 1 year after vaccination (r = 0.90, P = 0.02). In conclusion, in patients seronegative to measles who were revaccinated with measles vaccine after ABMT, a pre-existing T cell response correlated with an impaired B cell response, while pre-existing low-level IgG antibodies had no significant influence on the IgG titer rise. A sustained T cell response to measles antigen before vaccination may thus be one possible explanation for measles vaccine failure in ABMT patients.     

The 5D4 antibody (anti-cyclin D1/D2) related antigen: cytoplasmic staining is correlated to the progression of gastric cancer

In order to clarify the relationship between cyclin D1 and D2 (CD1/CD2) overexpression and progression, 191 gastric cancer cases (81 early and 110 advanced cancers) were investigated using the 5D4 monoclonal antibody for both CD1/CD2 in immunohistochemistry. 5D4 immunoreactivity was noted in 68 (35.6%) cases, staining being restricted to the nucleus in 27 (14.1%) cases, the cytoplasm in 34 (17.8%) cases, and its presence in both the nucleus and cytoplasm in seven (3.7%). Cases demonstrating cytoplasmic positivity, including both positive cases, were significantly more frequent in advanced cancers (P = 0.010), those having lymph node metastasis (P = 0.004) and cases showing cancer invasion of vessels (P = 0.009), although no relation to histological malignant grading was apparent. In contrast, cases of nuclear positivity behaved no differently from 5D4-negative cases. Statistics showed a trend where survival in patients was worse in the cytoplasm-positive cases than the cytoplasm-negative group. However, multivariate analysis revealed no independent statistical significance in the cytoplasmic positivity of prognosis. Additional studies using DCS-6 antibody for CD1 and C-17 antibody for CD2, suggest that nuclear staining of 5D4 indicates the presence of CD1 but cytoplasmic staining is derived from an antigen that is related to CD2. In conclusion, the present results indicate that the accumulation of CD2 in the cytoplasm may play some role in the progression of gastric cancers but not prognosis; however, CD1 overexpression is not linked to either.     

Kinetics of hepatitis B surface antigen-specific immune responses in acute and chronic hepatitis B or after HBs vaccination: stimulation of the in vitro antibody response by interferon gamma

Because cellular and humoral immune responses against the hepatitis B virus (HBV) surface antigen (HBs) might be crucial to overcome HBV infection, HBs-specific B- and T-cell responses of HBV patients and HBs vaccine recipients were analyzed quantitatively and functionally. In patients with acute hepatitis B (AHB), transient high anti-HBs-secreting B-cell frequencies were observed early after clinical onset, whereas 1 patient who probably developed chronic infection and chronic HBV carriers had absent or weak B- and T-cell responses. In HBs vaccine recipients, maximal HBs-specific B- and T-cell responses were detected after the first injection that decreased gradually before anti-HBs antibodies appeared in serum. Years after vaccination, anti-HBs-secreting B cells were enriched in the bone marrow. After in vitro stimulation with HBsAg, peripheral blood mononuclear cells (PBMC) of only 1 of 5 acute and 1 of 6 chronic HBV patients, but of all 6 vaccine recipients, secreted varying amounts of interferon gamma (IFN-gamma), but no interleukin-4 (IL-4) or IL-5. Furthermore, the addition of IFN-gamma, but not of IL-2, -4, -12, or IFN-alpha, resulted in strong increases of anti-HBs-secreting B cells in vaccine recipients and chronic carriers. In conclusion, circulating anti-HBs-secreting B cells were significantly higher in early acute hepatitis B or early after HBs vaccination than in chronic hepatitis B and decreased in the follow-up as a result of compartmentalization to lymphoid tissues. Release of IFN-gamma by antigen-stimulated T cells might be critical for anti-HBs formation.     

Prediction of response to hepatitis B vaccine in health care workers: whose titers of antibody to hepatitis B surface antigen should be determined after a three-dose series, and what are the implications in terms of cost-effectiveness?

We identified the demographics of 385 health care workers (HCWs) to identify those whose chance of developing a protective response to a standard primary hepatitis B immunization series was so high that the need for testing for antibodies to hepatitis B surface antigen (anti-HBs) would be obviated following immunization. In addition, using sensitivity analysis, we analyzed the economic consequences of not determining anti-HBs titers for any individual after primary immunization and of using the Centers for Disease Control and Prevention (CDC)-recommended post-hepatitis B exposure prophylaxis for high-risk HCWs. Nonsmoking women < 50 years old with a weight-height index of < 42 had a 98.2 +/- 0.9% chance of developing a protective anti-HBs titer. Male nonsmokers < 50 years old with a weight-height index of < 29 had a 94.7 +/- 1.8% chance of a protective response. Economic analysis revealed that use of the CDC guidelines for post-hepatitis B exposure prophylaxis in male HCWs whose anti-HBs status is unknown is always more cost-effective than determining anti-HBs titers following primary immunization for those at high risk. In female HCWs, post-hepatitis B exposure prophylaxis is more cost-effective until hepatitis B exposure rats are approximately 50%. It is possible to predict who will have a high probability of developing a protective response to hepatitis B vaccine; for these people, determining postimmunization anti-HBs titers is unnecessary and not cost-effective.