Developing an effective vaccine against HIV infection remains an urgent goal. We used a DNA prime/fowlpox virus boost regimen to immunize Chinese rhesus macaques. The animals were challenged intramuscularly with pathogenic molecularly cloned SHIV-KB9. Immunogenicity and protective efficacy of vaccines were investigated by measuring IFN-
Human immunodeficiency virus type 1 (HIV-1) is the aetiological agent of acquired immune deficiency syndrome (AIDS). Since the first case of HIV-1 infection was reported in Los Angeles in 1981 [
Undoubtedly, the best and most economical solution to eradicate or control the spread of HIV-1 is to develop safe and effective vaccine. Although considerable efforts have been devoted toward this goal, the success of available vaccines has not been demonstrated [
To further confirm the immunogenicity and protective effect of this vaccine, we immunized Chinese rhesus macaques with a DNA prime/fowlpox virus boost regimen. These animals were challenged intravenously with pathogenic molecularly cloned SHIV-KB9. The monkeys were monitored by measuring their IFN-
Chinese-origin rhesus macaques (Ch Rh), 3–5 years of age, both female and male, and with no signs of clinical diseases, were provided by the Laboratory Animal Center, Academy of Military Medical Sciences. Sixteen Chinese rhesus macaques from Guangxi, aged 3–5 years, weighing 3–5 kg, and without simian immunodeficiency virus (SIV), monkey T lymphocytes of I virus (STLV), monkey ART D-type virus (SRV/D), or B virus infection, were bred and provided by the experimental Animal Center of Military Medical Sciences. The present research project was approved by the relevant ethics review committee. Animal husbandry and sample collection were in accordance with relevant biosecurity requirements.
The vaccines used in the current study are recombinant DNA vaccine rDNA/pVMp24 and recombinant fowlpox virus rFPV/Mp24. Both are epitope-based vaccines containing the same immunogens, which includes a Kozak translation initiation sequence, ER signal peptide, 29 HIV dominant epitopes (24 CTL or CD8 T-cell epitopes and 5 B-cell epitopes), and HIV-1 p24 protein. The immunogens were provided by professor Ningyi Jin of the Institute of Military Veterinary Medicine, Academy of Military Medical Sciences. The schematic representation of the rDNA and rFPV vaccine constructs is shown in Figure
Schematic representation of the rDNA and rFPV vaccine constructs. The functional elements of the expression vector are the following. PCMV: human cytomegalovirus (CMV) immediate-early promoter/enhancer; Kozak: a Kozak translation-initiation sequence and an initiation codon (ATG) for proper initiation of translation; ER signal: endoplasmic reticulum signal peptide; MEG(4): multi-epitope gene (including 4 epitopes); P24: HIV-1 capsid protein; MEG(25): multi-epitope gene (including 25 epitopes); BGHpA: Bovine growth hormone (BGH) polyadenylation signal; TKL: the left recombinant fowlpox virus; PE/L: early and late promoter of fowlpox virus; T5NT: terminal signal of fowlpox virus; TKR: the right recombinant fowlpox virus.
The Chinese rhesus macaques were randomly divided into 2 groups (4 macaques per group). Each group was primed intramuscularly (i.m.) with rDNA/pVMp24 (500
The immunization schedule and routes of administration are outlined in Figure
Immunization and challenge schedule. rDNA: recombinant DNA; rFPV: recombinant FPV.
In the presence of EDTA anticoagulant, hind-limb venous blood was collected at 7, 13, 21, 28, and 35 d postinfection. The samples were sent to the laboratory within 6 h. Part of the unclotted blood was processed for blood routine examination and flow analysis. The remaining samples were centrifuged at 1700 rpm and kept at room temperature for 10 min. Plasma was analyzed for virus RNA load and antibodies. PBMC was separated from the blood cell for enzyme-linked immunospot (ELISPOT) and other analyses. Plasma was stored at −20°C, and the PBMC was frozen in liquid nitrogen.
ELISPOT assays were conducted to evaluate the gamma interferon-(IFN-
Sequence alignment indicates that HIV-1 p24 and SHIV p24 proteins have strong homology. Thus, the antibodies induced by the SHIV virus have a strong cross-immune response to the HIV antigen. In the present study, the HIV-1-specific antibody responses were measured by the third-generation total HIV-binding antibody diagnostic kit (Vironostika HIV Uni-Form II Plus O, BioMérieux Corporate, France). The experiment was conducted by employing the enzyme-linked immunosorbent assay according to the protocols of the manufacturer and the literature.
The plasma viral RNA was extracted by QIAGEN Viral RNA minikit (QIAGEN company) and analyzed by real-time PCR using TaqMan EZ RT-PCR Core Reagents kit (ABI Company) and ABI Prism 7700 apparatus. SHIV viral RNA in the samples was quantified by the standard curve derived from RNA standards.
We used three nonhuman primate antibodies, FITC-CD3, PerCP-CD4, and PE-CD8 (BD Pharmingen Inc.) in the flow analysis. Each flow tube contained 5
Differences between the groups were analyzed by Student’s
ELISPOT method was used to determine the IFN-
Number of IFN-
After the SHIV-KB9 virus attacks, all animals in the immunized group showed different degrees of ELISPOT-positive responses (peak in the range of 115–890 SFC/106 cells). At day 7 postinfection (29 w), a rapid increase in ELISPOT response was detected, and at day 21 (31 w), the ELISPOT response remained at an appropriate response level. These results suggest that the vaccine produced in the present study has good cellular memory immune response.
The antibody analysis results after infection are shown in Table
Whole-virus HIV-specific binding antibody titers after the challenge.
Groups | Animals | Whole-virus HIV-1 specific antibody titers | ||||
7 days | 14 days | 21 days | 28 days | 35 days | ||
Control | M1 | 0 | 0 | 0 | 0 | 1 |
M2 | 0 | 0 | 0 | 0 | 1 | |
M3 | 0 | 0 | 0 | 1 | 1 | |
M4 | 0 | 0 | 0 | 0 | 0 | |
Vaccine | M5 | 0 | 0 | 1 | 1 | 3 |
M6 | 0 | 0 | 0 | 1 | 2 | |
M7 | 0 | 0 | 1 | 1 | 2 | |
M8 | 0 | 0 | 1 | 2 | 3 |
The results of plasma viral RNA load are shown in Figure
Plasma viral load analysis post-SHIV-KB9 challenge. Viremia was quantified by RT-PCR. (a) Dynamics of viral load for each group. (b) Average value of viral load for each group.
Flow analysis of the T-lymphocyte subsets is shown in Figure
CD4/CD8 ratio analysis and total CD4 counts post-SHIV-KB9 challenge.
SIV/rhesus macaques are the most effective models for the investigation of the mechanisms for HIV pathogenesis and prevention [
Previous studies have shown that natural viral antigen may contain components with negative effects on protective responses including several immune suppression and immune pathological sequences. These components can interfere with the immune response and block the cell signaling pathway, leading to loss of balance for Th1/Th2 type immune response in the body [
“Prime-boost” immunization strategy is a sensational topic in current vaccine study [
In the study, we used an rDNA/rFPV “prime-boost” coimmunization strategy to immunize the Chinese rhesus macaques. Moreover, we used the SHIV-KB9 infection to analyze the immune protective effect of the vaccine. The results show that, during the primary immunization of rDNA/pVMp24 vaccine, the vaccine only induced a relatively low level of IFN-
IFN-
With regard to humoral immunity, we focused on the production rate of p24 antibodies, antibody titers, and antibody duration. The M1 and M2 in the negative control group showed weak HIV-1 binding antibody response after 35 days of virus attack. M3 showed positive response at days 28 and 35. However, the antibody titers did not rise. M4 remained negative during the experiment. Three animals in the immunized group (M5, M7, M8) showed positive antibody response at day 21, and the antibody titers exhibited a smooth, steady rise over time, indicating that the immunized group effectively induced the production of specific antibodies.
Viral load is a major parameter which can be used to evaluate whether HIV vaccine can induce immune protection, and the viral load change after the viral attack predicts the progress of the disease. In the present study, the positive response of viral load appeared in the control animals after 7 days of viral attack, and the peak value of average load occurred at day 13. In contrast, the peak value of the average load occurred at day 17 in the immunized group, and the peak value was lower than that in the control group. These results suggest that the vaccine delays the production of virus peak point at the early-infection stage and reduces the viral load at the peak point.
After virus infection, all the rhesus macaques in the negative control group exhibited the inversion phenomenon in CD4/CD8 ratio at day 13, and the ratio continued to decline rapidly. No recovery of CD4/CD8 ratio was observed during the experiment. However, in the immunized animals, the average CD4/CD8 ratio decreased at first, subsequently increased, and finally was restored to a relatively higher level at day 35. This indicates that the vaccine effectively induced T-cell proliferation.
The fowlpox virus expression system in the study is a newly developed poxvirus vector based on vaccinia virus vector [
This work was supported by Grants from the National 863 Project of China (no. 2006AA02Z447) and National Natural Science Foundation of China (no. 81001342).