Human cytomegalovirus (HCMV) glycoprotein complex II (gcII) consists of two glycoproteins, gM and gN. Although gcII specific IgG purified from HCMV positive patient sera can neutralize HCMV, there has been no report describing the generation of virus-neutralizing antibodies by immunization with individual recombinant gM or gN antigens. In the current study, gM and gN antigens were expressed by the mammalian expression vector pJW4303 and used as DNA vaccines to determine the immunogenicity of these proteins. Sera from mice or rabbits immunized with individual or combinations of gM and gN DNA vaccines contained gM and gN specific antibodies as confirmed by ELISA and Western blot analyses. The combined gM and gN antigens induced the strongest antibody responses that recognized both gM and gcII complex while gM DNA vaccine alone could only elicit antibody specific for gM antigen. When given alone, the gN DNA vaccine did not induce detectable gcII specific antibody even though in vitro gN expression was confirmed by the formation of gM/gN complex in FSK cells using a gN-specific monoclonal antibody 14-16A. The neutralizing antibody titer of anti-gM/gN sera (1:128) was higher than that of anti-gM sera (1:32) against the autologous virus, HCMV AD169. Heterologous HCMV strains including Towne and Davis could also be neutralized by the anti-gM/gN antisera. Our data supported the rationale for the use of the HCMV gM/gN protein complex as protective antigens for subunit based HCMV vaccine development. DNA vaccination is an effective approach to express the gM/gN antigen complex in vivo without the need to express and purify these highly insoluble and structurally complicated antigens.
[Show abstract][Hide abstract] ABSTRACT: Human cytomegalovirus (HCMV) is a ubiquitous pathogen that causes serious problems in immunocompromised or immunologically immature hosts. Vaccination is the preferred approach for prevention of HCMV infection, but so far no approved HCMV vaccine is available. In this study, we assessed the immunogenicity and protective immunity of a formalin-inactivated murine cytomegalovirus vaccine (FI-MCMV) in a mouse model in combination with adjuvants MF59, alum, or chitosan.
Specific-pathogen-free BALB/c mice aged 6-8 weeks were immunized twice, 3 weeks apart, with various doses of FI-MCMV (0.25 mug, 1 mug, 4 mug) with or without adjuvant. Mice were challenged with a lethal dose (5 x LD50) of a more virulent mouse salivary gland-passaged MCMV 3 weeks after the second immunization. The protective immunity of the vaccine was evaluated by determining the survival rates, residual spleen and salivary gland viral loads, body weight changes, and serum anti-MCMV IgG titers.
Immunization with FI-MCMV vaccine induced a high level of specific antibody response. Antigen sparing was achieved by the addition of an adjuvant, which significantly enhanced the humoral response to vaccine antigens with a wide range of doses. The level of live virus detected in the spleen on day 5 and in the salivary glands on day 21 after the lethal challenge was significantly lower in adjuvant-treated groups than in controls. Survival rates in adjuvant-treated groups also increased significantly. Furthermore, these protective immune responses were sustained for at least 6 months following immunization.
These results show that inactivated MCMV vaccine is effective, and that the adjuvanted FI-MCMV vaccine provides more effective and longer-term protection than the adjuvant-free vaccine.
"The gN and gO genes are encoded in adjacent loci on the genome, consistent with their observed linkage (Mattick et al., 2004). They also have linked functions in secondary envelopment, assembly and exit of the virus and antibodies can neutralise infection suggesting key roles for pathogenicity (Jiang et al., 2008; Krzyzaniak et al., 2007; Mattick et al., 2004; Paterson et al., 2002; Rasmussen et al., 2002; Shen et al., 2007). They are amongst a select subset of hypervariable genes which are suited to following molecular genotyping, whereas earlier studies using less variable loci such as gB and gH are more difficult to interpret. "
[Show abstract][Hide abstract] ABSTRACT: Human cytomegalovirus, HCMV, was analysed using real-time quantitative PCR in symptomatic or asymptomatic pediatric cohorts from HIV-1 infected, exposed (HIV-1+ mothers), or uninfected groups in Zambia, an HIV-1/AIDS endemic region of Africa. HCMV infections were identified in 94% samples from HIV-1+ respiratory pediatric mortalities, 50% with high DNA loads of 10(3)-10(8) copies/10(6) cells. In comparison, HCMV viremia with high DNA loads, indicative of acute infections, were in 10% hospitalised febrile infants, with 50% HIV-1+. Whereas high sera loads were in 1% of asymptomatic infants, with 2% HIV-1+, and higher levels in both HIV-1 infected or exposed, but negative infants. All 8 linked-hypervariable glycoprotein gN-gO genotypes were shown, including identification of a new gN4d group with gO5 linkage (previously only Merlin reference strain), and samples with multiple infections. Overall, this shows global genotypes in Africa (unlike some herpesviruses) and acute pediatric HCMV infections in both HIV-1+ plus exposed, but uninfected infants, an emerging group.
[Show abstract][Hide abstract] ABSTRACT: Although infection with human cytomegalovirus (HCMV) is ubiquitous and usually asymptomatic, there are individuals at high risk for serious HCMV disease. These include solid organ and hematopoietic stem cell (HSC) transplant patients, individuals with HIV infection, and the fetus. Since immunity to HCMV ameliorates the severity of disease, there have been efforts made for over 30 years to develop vaccines for use in these high-risk settings. However, in spite of these efforts, no HCMV vaccine appears to be approaching imminent licensure. The reasons for the failure to achieve the goal of a licensed HCMV vaccine are complex, but several key problems stand out. First, the host immune correlates of protective immunity are not yet clear. Secondly, the viral proteins that should be included in a HCMV vaccine are uncertain. Third, clinical trials have largely focused on immunocompromised patients, a population that may not be relevant to the problem of protection of the fetus against congenital infection. Fourth, the ultimate target population for HCMV vaccination remains unclear. Finally, and most importantly, there has been insufficient education about the problem of HCMV infection, particularly among women of child-bearing age and in the lay public. This review considers the strategies that have been explored to date in development of HCMV vaccines, and summarizes both active clinical trials as well as novel technologies that merit future consideration toward the goal of prevention of this significant public health problem.
Current topics in microbiology and immunology 01/2008; 325:361-82. DOI:10.1007/978-3-540-77349-8_20 · 4.10 Impact Factor
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