Genetic Stability, Biodistribution And Humoral Immunity Studies Of A Combined Therapeutic HIV Vaccine Candidate Ad5/35-HGEC And MVA-HGEC

Genetic stability, biodistribution and humoral immunity studies of a combined therapeutic HIV vaccine candidate Ad5/35-HGEC and MVA-HGECGiven the significant threat of human immunodeficiency virus (HIV) infection worldwide, an ideal vaccine capable of preventing HIV infection as well as providing sterilizing immunity is urgently needed to control the global AIDS pandemic. A vaccine that elicits both specific antibodies and cell-mediated immunity (CTL) is regarded to protect against HIV efficiently. Among the most promising approaches to induce such complex immunity are heterologous prime-boost vaccination regimens, in particular ones containing live viral vector. Therefore, a prime-boost strategy was developed with a replication-defective chimeric Ad5 vector with Ad type 35 fiber (Ad5/35-HGEC) and a recombinant MVA vector (MVA-HGEC), both of which encode HIV-1 clade C gag and gp160 genes.The purpose of this study was to investigate the genetic stability and biodistribution of the recombinant viral vectors. Meanwhile, humoral immunity against HIV conferred by Ad5/35-HGEC prime and MVA-HGEC boost vaccination was evaluated.Chapter One: Genetic stability investigation of Ad5/35-HGEC and MVA-HGECIn, this part, genetic stability of recombinant viral vectors was investigated regarding of the safety and efficiency. Ad5/35-HGEC was passaged in 293 cells while MVA-HGEC was passaged in CEF cells from generation 5 to 20. Then PCR and DNA sequencing methods were used to check sequence fidelity of gag and gp160 genes within 20 generation, and no changes were found. Meanwhile, recombinant viral vectors were transfected with 293 cells, and Western blot was performed to evaluate the stability of protein expression in different generations. It was confirmed that target protein, including gag and gp160, could express in both vectors within 20 passages, importantly, no significant difference was found.Chapter Two: Biodistribution study of Ad5/35-HGEC and MVA-HGEC with TaqMan real-time PCR method In this part, a TaqMan real-time PCR method was used to track the time-dependent distribution of the vectors in vivo. This technique was shown to be rapid, specific, reproducible and highly sensitive.It was found that Ad5/35-HGEC remained mainly in local muscle tissues at injection sites post intramuscular (i.m) administration to Balb/c mice at 1.6×109vp dosage, some was detected in liver, heart and lung, while little was found in blood and other tissues. 1 week post administration the concentration of Ad5/35 and gag was 4332.6±2562.9 copies?mg-1 and 1472.3±1053.8copies?mg-1, respectively. However, 8 weeks later, viral DNAs could hardly be detected within any tissues.Interestingly, MVA-HGEC also remained mainly at injection sites with a 2×107pfu dosage. The concentrations of MVA and gag were, respectively, 859.5±257.2 copies?mg-1and 1120.6±713.7 0.5 hour post administration. However, little could be detected 3 days later, suggesting a rapid degradation and clearance.Besides, the risk of insertional mutagenesis or germline transmission of viral vectors was evaluated by checking the level of viral DNAs in muscle and gonad. It was found that viral DNA concentrations in muscle decreased quickly and couldn't be detected at last, while viral DNAs could hardly be detected in gonad. These results suggest a low probability of toxicity and satisfied safety of the vectors used.Chapter Three: Expression distribution study of Ad5/35-Luc with IVISIn this part, in vivo imaging system (IVIS) was utilized to provide a visual result for the expression distribution of Ad5/35 vector in animals.To achieve this goal, Ad5/35 vector was labeled with luciferase gene. Bioluminence was detected with IVIS on days 1, 3, 7, 10, 14, 21, 28 and 35 post i.m. administration at a dosage of 2×10¹⁰vp. It was showed that luciferase expression was found mainly at injection sites with the highest level at day 7, lasting for a 28-day period, while little bioluminence was obtained from other sites. These results were similar to that obtained with real-time PCR method.Chapter Four: Humoral immunity study of Ad5/35-HGEC prime and MVA-HGEC boost vaccinationIn this study, a specific antibody against HIV-1 C p24 was detected by an enzyme-linked immunosorbent assay (ELISA) post Ad5/35-HGEC prime and MVA-HGEC boost vaccination in non-human primates.Rhesus monkeys with HIV-1 C serum negative were divided into three groups, one administrated with normal sodium was used as negative group, the other two groups were administrated with vectors with or without HIV genes, respectively. Absorbance was measured at a wavelength of 450 nm by using a Microplate reader, and the cut-off (CO) value was defined as that value which exceeded the mean OD450 of the negative group by three standard deviations (Mean+3 Std). The CO value was 0.389 here. When the value of OD450/CO exceeds 1, the sample was considered positive, otherwise it was judged as negative.According to this value, a detectable HIV-specific serum Ab response developed within 1 week and achieved its peak (OD450=0.857±0.230)at week 6th week post prime injection with Ad5/35-HGEC at 1×1011vp dosage. The specific response peaked again(OD450=1.025±0.123)4 weeks after the boost at 8th week with MVA-HGEC at 1×10⁸ pfu dosage. Significant differences between the negative and vaccine groups could be found by analysis of variance (ANOVA).However, all of the OD450 values from the vaccine group were less than the CO value after 50-fold dilutions. That is to say, the titers of serum Ab elicited by the HIV vaccine were less than 1:50. Thus, improvement of regimen should be made for a strong humoral immunity.In conclusion, both vectors display good genetic stability within 20 generation. Evaluation of biodistribution and humoral immunity suggests their safety and efficiency post administration into animals. An important outcome from the present study is that this prime-boost vaccine regimen is a promising candidate for use as an HIV vaccine.