Introduction

Despite the fact that human immunodeficiency virus-1 (HIV-1) was discovered more than 30 years ago, there is an urgent need for a safe and effective global HIV-1 vaccine. While HIV-1 vaccine clinical efficacy trials have not been stopped, only four vaccine concepts have been evaluated for protective efficacy in humans [1, 2]. However, the tested vaccines ultimately failed to provide sufficient protection against HIV infection in clinical trials [3]. One of the lessons learned from these failures was that HIV-1 vaccines should be evaluated first in stringent preclinical studies [2].

Fowlpox virus (FPV) has been used as a live vector vaccines against infectious diseases and cancer in poultry, humans, and other mammals, and has been shown to be safe [4,5,6]. FPV undergoes a transient infection in which infection by progeny virus does not occur in mammalian cells, but the virus and target genes are still expressed in these cells. Thus, FPV is likely to be a promising vector for delivering HIV-1 antigen in a vaccine.

Prior clinical trials have provided insights regarding the optimal HIV-1 antigens for use in a vaccine. The RV144 HIV-1 vaccine study demonstrated a vaccine efficacy of 31.2% compared to placebo in Thailand [7], which was directly correlated with variable regions 1 and 2 (V1V2) of the HIV-1 envelope (Env) IgG antibodies and Env-specific IgA antibodies that were elicited [8]. This result suggested that Env proteins could be used to elicit broadly neutralizing antibodies against HIV-1 and/or simian immunodeficiency virus (SIV) [9,10,11]. However, broadly neutralizing antibodies are difficult to induce because of their unique characteristics [12]. Therefore, an alternative approach is to elicit antibodies that are similar to neutralizing antibodies and able to neutralize many HIV-1 strains [13,14,15,16]. In addition to Env, HIV-1 gag and SIV gag can also elicit high frequencies of gag-specific CD8+ T cells and a long-lasting humoral response [17,18,19]. Therefore, the gag and env proteins would likely be the preferred antigen for use in a recombinant FPV (rFPV) vaccine candidate.

As previously noted, HIV-1 vaccine candidates should be extensively studied in stringent preclinical models prior to human efficacy studies. SIV infection in rhesus macaque monkeys is one such system which has been used for evaluating HIV-1 vaccine. Thus, the first step in developing an rFPV HIV-1 vaccine, is developing an rFPV vaccine expressing SIV antigens that can be experimentally tested in the preclinical models to estimate the immunogenicity and protective efficacy of the SIV/HIV vaccine candidates. However, the macaque model is limited in that the experiments are expensive, use outbred animals, and use relatively small numbers of animals. Therefore, the immunogenicity of candidate vaccines are often tested in small animal models prior to primate studies.

Therefore, in this study we constructed an rFPV vaccine expressing the SIV gag and SIV envT genes to evaluate the immunogenicity of the rFPV-gag/env vaccine in BALB/c mice to provide a foundation for evaluating the vaccine construct in the macaque model.

Materials and Methods

Plasmid, Virus, Cells and Animals

pVR-SIV gag and pVR-SIV envT were kindly provided by Xia Feng at the Chinese Center for Disease Control and Prevention. The gag and envT genes belong to the SIV/mac239 (Genebank accession #M33262), envT was constructed by truncating env.

The rFPV vaccine was constructed from the Chinese FPV vaccine strain FPV282E4 and an FPV shuttle vector. The plasmid pVAX-Cre and FPV shuttle vectors were constructed previously. FPV282E4 was purchased from the Animal Pharmaceutical Factory of Nanjing (Nanjing, China). Baby hamster kidney (BHK) cells were cultured in DMEM with 10% fetal bovine serum and 1% penicillin (10,000 U/mL) / streptomycin (10,000 μg/mL) solution. Eight-day-old specific-pathogen free chickens were used to prepare the chick embryo fibroblast (CEF) cells that were purchased from Meiliyaweitong Experimental Animal Technology Co. Ltd (Beijing, China).

Six-week-old, female BALB/c mice (Experimental Animal Center, Academy of Military Medical Sciences of PLA, Beijing, China) were housed in an animal facility.

Construction of Recombinant Plasmids

The FPV shuttle vector pT3eGFP150 (4816 bp) containing the left (TKL) and right (TKR) halves of the TK gene, a double-gene expression cassette, and EGFP reporter gene was constructed in our laboratory [20]. The 1.5 kb SIV gag and 2.1 kb SIV envT genes were amplified and cloned into the multiple cloning site (MCS) 1 and MCS 2 respectively, to produce pT3eGFP150-SIV gag-SIV envT (pT3eGFP–Sg–Se).

Isolation of rFPVSg–Se from CEF Cells

CEF cells were infected with FPV282E4 at a multiplicity of infection (MOI) of 1 for 2 h. The cells were then transfected with 1 μg of the plasmid pT3eGFP150–Sg–Se using Lipofectamine 2000 (Invitrogen, US). The infected cells expressing EGFP were picked out under a fluorescence microscope and used for additional rounds of infection. After 12 rounds of plaque screening, the purified virus was termed rFPVSg–Se. rFPVSg–Se was then characterized by PCR, RT-PCR, and Western-blot.

Characterization of rFPVSg–Se

The genomic DNA (gDNA) and total RNA from cells infected with rFPVSg–Se were extracted to use as templates for amplifying the SIV gag, SIV envT, FPV-P4b, and TK genes by PCR and RT-PCR. The PCR reaction conditions were 95 °C 5 min, 30 cycles of 95 °C 30 s, 60 °C 30 s and 72 °C 2 min 10 s, and a final extension at 72 °C for 10 min. The primers utilized are shown in Table 1. The SIV gag and SIV envT genes were inserted into the FPV genome such that the TK gene was broken and blocked, therefore the TK gene was used as a selection marker to purify rFPVSg–Se. The P4b gene encoding the virion nucleoprotein (75 kDa), which is widely found in FPV, was used to identify FPV [21].

Table 1 Primer sequences used for PCR and the expected lengths of the amplified fragments
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Considering the characteristics of FPV, we need to test the target proteins whether could be expressed effectively in rFPVSg–Se-infected BHK cells. Therefore, BHK cells were infected with rFPVSg–Se at an MOI of 5 for 72 h, and total protein was extracted from rFPVSg–Se-infected cells to probe for expression of the SIV gag and SIV envT proteins by Western-blot. A rabbit anti-p27 (SIV/mac 239) antibody, rabbit anti-gp120/160 (SIV/mac239) antibody, and mouse anti-β-actin antibody were used as the primary antibodies to identify the SIV gag, SIV envT and β-actin proteins, respectively. HRP-conjugated goat anti-rabbit lgG and goat anti-mouse IgG were used as secondary antibodies. FPV-infected cells were used as the negative control, and β-actin served as the internal control.

To assess the stability of the SIV gag and SIV envT insertions, rFPVSg–Se was passaged 20 times, and the genomic DNA (gDNA), RNA, and total protein were extracted from the 1st, 5th, 10th, 15th, and 20th passages at the genetic (PCR, RT-PCR) and protein (Western-blot) levels.

Single Immunization

Fifty-four (54) female BABL/c mice were divided evenly into three groups (n = 18). One group was immunized with 1 × 106 plaque forming units (PFU) of rFPVSg–Se (rFPVSg–Se) by the intramuscular route, one group received 1 × 106 PFU of FPV282E4 (FPV282E4), and one received 100 μL of PBS (PBS). Blood samples were harvested at 1, 7, 14, 21, 28, and 35 days post immunization. The serum was collected and stored at − 80 °C until it was used in ELISAs to detect the levels of SIV- and vector-specific antibodies. The experimental design is shown in Fig. 2a.

Prime-Boost Immunization

Seventy-two (72) female BALB/c mice were divided into three experimental groups (n = 24). Animals in the rFPVSg–Se group were primed with 1 × 107 PFU of rFPVSg–Se in 100 μL of PBS by the intramuscular route at day 0 and were boosted with the same dosage at day 21. The second group received 1 × 106 PFU of FPV282E4 (FPV282E4) at days 0 and 21, and the third group received 100 μL of PBS on days 0 and 21. Serum sample were collected at days 1, 7, 14, 21, 28, 35, 42, and 49 post immunization to detect SIV and vector-specific antibodies by ELISA. The experimental design is shown in Fig. 3a.

ELISA Analysis and Residual Virus Detection

SIV-specific antibody responses to heterologous SIVmac239 gag and gp120 (env) were detected by ELISA. For the ELISA, the SIV gag and gp120 proteins were utilized as capture antibodies, serum samples from immunized mice were the primary antibody (diluted in 1:20 in PBS) added to the wells, and a peroxidase-conjugated goat anti-mouse IgG antibody was used as the second antibody (diluted 3000-fold in PBS), and the optical density (OD) was detected at 492 nm. A standard curve was constructed using the same approach. Vector-specific antibodies against inactivated FPV282E4 (1 × 106 PFU/ 96 well plate) were detected in the same manner.

gDNA was isolated from the hearts, livers, spleens, lungs, kidneys, brains, and muscles of mice primed with rFPVSg–Se on days 1, 3, 5, 7, and 14 post immunization for use as templates for amplifying the SIV gag, SIV envT, and P4b genes to detect residual rFPVSg–Se. The primers utilized are shown in Table 1.

Statistical Analysis

Statistical analyses were performed using the Graphpad Prism software version 5.0 (San Diego, CA, USA). Differences between groups with a p value < 0.05 were considered to be statistically significant. Data are presented as the mean ± standard deviation (SD).

Results

Construction and Characterization of the rFPVsg–se Vaccine

To construct rFPVsg–se, we first generated the recombinant plasmid pT3eGFP150–Sg–Se (8509 bp) shown in Fig. 1a. The successful construction of pT3eGFP150–Sg–Se was confirmed by restriction enzyme digestion using Afl II/Not I to identify the SIV gag insert (1533 bp) and Kpn I/Sal I to identify the SIV envT insert (2160 bp) as shown in Fig. 1b.

Fig. 1
figure 1

Construction and characterization of the rFPVsg–se vaccine. a Schematic diagram of the strucutre of the plasmid pT3eGFP150–Sg–Se. b Identification of pT3eGFP150–Sg–Se by restriction enzyme digestion. c EGFP expression was used to screen for cells infected with rFPVSg–Se under a fluorescent microscope at 72 h post-transfection, and after 4, 10, or 12 rounds of plaque screening. d Identification of rFPVSg–Se by PCR. e Western bolt analysis of SIV gag and SIV envT in rFPVSg–Se-infected CEF cells compared to mock infection. f Western bolt analysis of SIV gag and SIV envT in rFPVSg–Se-infected BHK cells compared to mock infection. g The SIV gag and SIV envT genes were amplified from the gDNA and cDNA of rFPVSg–Se-infected CEF cells every five passage (1, 5, 10, 15 and 20) for twenty passages. h Protein expression levels of SIV gag and SIV envT were assessed by Western-blotting every five passages for twenty passages

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Having established the SIV genes were correctly inserted, we then infected CEF cells with FPV282E4 and transfected the infected cells with pT3eGFP150–Sg–Se. The rFPVSg–Se-infected cells were then selected based on expression of the EGFP reporter gene. Rounds of selection for rFPVSg–Se continued until all of the plaques expressed EGFP (Fig. 1c). The SIV gag (1503 bp), SIV envT (2106 bp), P4b (578 bp), and TK (1006 bp) genes were amplified from rFPV-sg–se infected cells by PCR and RT-PCR (Fig. 1d), indicating that the foreign genes were integrated into the rFPVSg–Se genome and transcribed. A pure stock of rFPVSg–Se, lacking contamination from FPV282E4, was obtained when the TK gene could not be amplified from gDNA or cDNA. The target proteins SIV gag (56 kDa) and SIV envT (120 kDa) could be identified in rFPVSg–Se-infected CEF and BHK cells by Western-blot (Fig. 1e, f).

To assess the stability of SIV gene expression, gDNA, RNA, and total protein was collected from rFPVSg–Se infected cells every five passages for 20 passages to assess the integration, transcriptional, and translational levels of SIV gag and SIV envT by PCR, RT-PCR (Fig. 1g) and Western-blot (Fig. 1h). The results demonstrated that rFPVSg–Se could be passaged at least 20 times and continue to stably express the inserted SIV genes.

Evaluating the Titer of SIV and FPV Specific Antibodies Elicited by Immunization with rFPVSg–Se

The ability of rFPVSg–Se to elicit antibodies targeting SIV gag and gp120 was first assessed following a single immunization (Fig. 2a). The levels of SIV- and FPV specific IgG antibodies in the serum of immunized mice were measured by ELISA at 1, 7, 14, 21, 28, and 35 days post immunization. Following a single immunization, the levels of SIV gag- and gp120-specific antibodies in the rFPVSg–Se immunized group were significantly higher than the other groups (p < 0.05, Fig. 2b, c). Vector-specific antibody levels were also markedly increased at day 7 post immunization in the rFPVSg–Se and FPV groups, but plateaued from 14 to 35 days post immunization (Fig. 2d).

Fig. 2
figure 2

Quantifying the serum antigen specific IgG titer in BALB/c mice following a single rFPVsg–se immunization. a Schematic of the experimental design. Female BALB/c mice were used (n = 18/group). The mice were divided into three groups and immunized with rFPVsg–se, FPV282E4, or PBS. b SIV gag-specific IgG titer, c SIV gp120-specific IgG titer, and d vector-specific antibody levels were measured in the serum by ELISA

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To test whether the antibody response could be boosted, we immunized mice twice on days 0 and 21. The serum antigen specific IgG titers were measured by ELISA on days 1, 7, 14, 21, 28, 35, 42, and 49 following the priming immunization (Fig. 3A). The levels of IgG antibodies specific to the SIV gag and gp120 proteins were further increased after boosting (p < 0.01, Fig. 3b, c). In contrast, the titer of vector-specific antibodies remained relatively stable after boosting (Fig. 3d). These results indicated that immunizing BALB/c mice with SIV gag and SIV gp120 in the FPV vector elicited an SIV-specific humoral response, and that a prime-boost strategy elicited higher titers of antibody than a single immunization.

Fig. 3
figure 3

Quantifying the serum antigen specific IgG titer in BALB/c mice following a prime-boost rFPVsg–se immunization. a Schematic of the experimental design. Female BALB/c mice were used (n = 24/group). The mice were divided into three groups and immunized with rFPVsg–se, FPV282E4, or PBS. b SIV gag-specific IgG titers, c SIV gp120-specific IgG titers, and d vector-specific antibody levels were measured in the serum by ELISA

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Detection of Residual rFPVSg–Se in Multiple Organs

As a safety measure and to demonstrate the transient nature of rFPVSg–Se infection, residual rFPVSg–Se was detected by amplifying the SIV gag, SIV envT, and p4b genes from the tissues and organs of rFPVSg–Se-immunized mice at days 1, 3, 5, 7 and 14 post immunization. rFPVSg–Se detection are shown in Fig. 4a, e, respectively. rFPVSg–Se was only detected in the muscle at 1, 3, 5, and 7 days post immunization. The preliminary results suggest that gene expression from 1 × 106 PFU of rFPVSg–Se was cleared from the injection site by at most 14 days post immunization in BABL/c mice.

Fig. 4
figure 4

Detection of residual virus in organs of rFPVSg–Se-immunized mice. Residual rFPVSg–Se was detected by PCR in the heart, liver, spleen, lung, kidney, brain, and muscle of immunized mice at a 1, b 3, c 5, d 7 and e 14 days post immunization

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Discussion

To successfully elicit a durable, long-lasting humoral response to protect against HIV-1 and SIV infection over many years multiple immunizations are necessary, which may lead to immune tolerance if the same vaccine is used every time. Therefore, multiple vaccine platforms are necessary to use in combined immunization strategies to prevent HIV and SIV infection over many years.

To make the rFPVSg–Se vaccine candidate tested in this study, we selected the gag and env genes which are well known to elicit a strong humoral and neutralizing antibody response in humans [22]. In early studies from our laboratory, BrdU selection has been used to construct recombinant vaccinia virus and rFPV vectors, this method generally has a low recombination efficiency, is labor intensive, and returns a high number of non-recombinant viruses during screening. Thus, we chose to use the EGFP reporter gene to construct the rFPV plasmid pT3eGFP150–Sg–Se. This method had the advantages that EGFP was only expressed in the recombinant viruses, and the screening process was visual based on easily observable green fluorescent plaques.

Immunization with rFPVSg–Se elicited an antigen specific IgG response to SIVmac239 gag and gp120 proteins using a prime-boost strategy. We also observed an antibody response to the FPV vector, which did not increase following the booster immunization. These results indicated that vaccination using rFPVsg–se could be repeated at least once to increase antibody titer.

While we have shown that high levels of SIV-specific antibodies can be elicited by rFPVSg–Se, future studies are required to characterize the neutralizing antibody titer, cellular immune response, and protective efficacy of the vaccine. In addition, rFPVSg–Se was not detected in the other organs by PCR, it is possible that rFPV does not bind to organs away from the injection site or that the low number of adsorbed-rFPVSg–Se virions is not sufficient to detect by PCR. Therefore, we also need to further verify that rFPVsg–se is not widely disseminated and is transiently infecting the recipient. Our work did show that SIV gag- and SIV envT-specific antibodies were elicited strongly by rFPVSg–Se in the BALB/c mouse model and provided a foundation for testing the immunogenicity and safety research of SIV/HIV rFPV candidate vaccines.