Role of complement and antibodies in controlling infection with pathogenic simian immunodeficiency virus (SIV) in macaques vaccinated with replication-deficient viral vectors
© Falkensammer et al; licensee BioMed Central Ltd. 2009
Received: 12 March 2009
Accepted: 21 June 2009
Published: 21 June 2009
We investigated the interplay between complement and antibodies upon priming with single-cycle replicating viral vectors (SCIV) encoding SIV antigens combined with Adeno5-SIV or SCIV pseudotyped with murine leukemia virus envelope boosting strategies. The vaccine was applied via spray-immunization to the tonsils of rhesus macaques and compared with systemic regimens.
Independent of the application regimen or route, viral loads were significantly reduced after challenge with SIVmac239 (p < 0.03) compared to controls. Considerable amounts of neutralizing antibodies were induced in systemic immunized monkeys. Most of the sera harvested during peak viremia exhibited a trend with an inverse correlation between complement C3-deposition on viral particles and plasma viral load within the different vaccination groups. In contrast, the amount of the observed complement-mediated lysis did not correlate with the reduction of SIV titres.
The heterologous prime-boost strategy with replication-deficient viral vectors administered exclusively via the tonsils did not induce any neutralizing antibodies before challenge. However, after challenge, comparable SIV-specific humoral immune responses were observed in all vaccinated animals. Immunization with single cycle immunodeficiency viruses mounts humoral immune responses comparable to live-attenuated immunodeficiency virus vaccines.
Beside cellular immune responses, humoral immunity is considered a key component in AIDS vaccine development. Already during early stages of viral infection, anti-envelope (env) antibodies (Abs) are thought to reduce viremia [1–3]. Their effector functions are still not completely defined. Some of such neutralizing antibodies (nAbs) may inhibit viral entry either by interfering with structures of the gp120/gp41 complex  or with env-epitopes that bind to chemokine receptors. Alternatively, they may cross-link virus particles and induce clearance of immune-complexed viruses by phagocytosis. Additionally, antibody dependent cellular cytotoxicity (ADCC) is thought to appear early during acute infection  and can also be detected at later stages of disease progression. ADCC has been studied in the SIV monkey model, was associated with the control of HIV in infected humans [6–8] and may contribute to a slower disease progression in long-term non-progressors .
A further arm of the humoral immune response is the complement system as an important mechanism of innate immune defence. Complement (C) has been shown to enhance the activity of nAbs . In synergy to the binding of Abs to viruses, C3 deposition, opsonization and immune complex formation are suggested to contribute to reduced viral infection rates. There is evidence that C-mediated lysis contributes mainly at early stages of HIV-1 infection to viremia control [11–13].
A major focus of current research is the design of safe and efficient vaccines providing a high level of protection against HIV. A promising approach is the application of replication-deficient single-cycle immunodeficiency viruses (SCIV) [14, 15]. Upon application, these viral constructs undergo only one single round of replication resulting in the production of non-infectious virus-like particles in vivo. The induced immune response is thought to protect from challenge by clearing infected cells.
A non-invasive application of live-attenuated SIV vaccines to the mucosa via the tonsils has been established. This approach induced protection against challenge with homologous SIV and SHIV, a SIV/HIV-1 hybridvirus containing HIV-1 envelope in the SIV backbone [16, 17]. Although effective, the delivery of attenuated retroviruses is not feasible in humans due to safety concerns [18, 19]. Thus, we adopted a heterologous prime-boost regimen through priming with SCIV and boosting with Adeno5 (Ad5)-SIV or SCIV. The vectors were either given systemically or exclusively mucosally.
weeks post immunization
1.8 × 109, a
1.2 × 108, a
1 × 1011, b
1 × 1011, b
2 × 109, a
6 × 1011, b
2 × 109, a
3 × 107, a
1 × 1011, c
2 × 1011, c
6 × 1011, c
The results of the systemic spread of SCIV after oral immunization, as well as analyses concerning the cellular immune responses, immunohistochemical and in situ hybridisation assays have been recently published by Stahl-Hennig et al. . In the present study, we characterized the humoral immune response in immunized and challenged rhesus macaques and investigated the contribution of the induced neutralizing and non-neutralizing antibodies, C-deposition on the viral surface and C-mediated lysis with regard to the control of retroviral infection.
Viral load levels
SIV neutralizing antibodies
By a yield reduction assay using SIVmac251, the first detectable nAbs were measurable in group 2 and 3 with mean fold inhibitions of 171.8 and 110.5, respectively, 4 weeks after the first boost (12 wpi). In group 1, nAbs remained undetectable upon immunization. However, after challenge with pathogenic SIVmac239, nAbs rapidly increased, and by 8 wpc these monkeys had increased nAb yields compared to cohort 2 and 3. After challenge, mean nAbs of control monkeys rose continuously, reaching the maximum mean fold inhibition of 499.0 at 20 wpc. At the end of the observation period (28 wpc) cohort 1, 2 and 3 developed maximum mean fold inhibition of 733.3, 572.8 and 523.8, respectively.
SIV env-specific IgG
Induction of complement-mediated lysis
%lysis day of challenge
%lysis 2 wpc
viral load 2 wpca
%lysis 28 wpc
viral load 28 wpca
2.7 × 103
1.8 × 103
4.7 × 104
6.6 × 103
1.3 × 104
3.1 × 103
6.6 × 104
3.4 × 104
1.9 × 103
6.9 × 104
1.1 × 104
1.2 × 103
2.1 × 104
6.8 × 102
1.9 × 104
1.8 × 105
5.5 × 105
4.1 × 103
2.4 × 105
2.4 × 104
4.3 × 104
7.7 × 102
9.0 × 105
1.2 × 105
6.7 × 106
1.6 × 106
2.1 × 106
1.4 × 104
1.0 × 106
4.2 × 104
Virus capture assay
During chronic infection, the C3 opsonization was more pronounced when compared to the C3-deposition induced by sera collected during the peak viremia. However, the correlation between C3-deposition and viral load was no longer observable (data not shown).
In this study we analyzed the efficacy of humoral immune responses induced by different vaccination strategies either combining a SCIV [VSV-G] prime with an adenoviral boost or administering SCIV only (Table 1). The used SCIV [VSV-G] vaccine provides a safer immunization strategy when compared to live-attenuated vaccines, as no replication-competent particles are generated . Adenoviral vectors have been used in the past, but were usually applied intramuscularly  and not via the tonsils. Although our approach did not induce sterilizing immunity, the vaccinated animals had a significantly reduced peak viremia after challenge with the highly pathogenic SIVmac239 when compared to the non-immunized but infected control animals. Peak viral load levels were reduced between 1 log in group 3 and 2 log in groups 1 and 2 (Figure 1A) . Similar reductions in the viral titre were achieved by an iv prime-boost strategy using SCIV as a vaccine . As many studies have emphasised that the long-term prognosis is significantly improved the lower the peak viral load levels are [24, 25], the decrease of the viral load by oral administration of our vaccine may provide profound benefit.
Along with the nAb titres, the levels of the total env-specific IgG were weak but mainly detectable in the systemically immunized animals of group 2 already 12 wpi. The detection of the Abs by FACS analysis using SIV-infected cells allows the detection of native, in vivo accessible epitopes only and may be less sensitive compared to ELISA detection systems. Stahl-Hennig et al.  used a gp130 ELISA with proteins expressed in E. coli for this animal study. However, these proteins do not reflect the in vivo conformation of the env-protein complex and may thus account for overestimated IgG titres and explain the controversial findings reported previously . It is possible that neutralizing antibodies are not detected by FACS, but will be recognized in ELISA assays. One example is the monoclonal antibody 2F5  which binds to the membrane proximal external region of gp41 during the fusion process but not in the native state. After infection with SIVmac239, the overall IgG response was dramatically boosted in all animals and ran parallel to the induction of nAbs. Interestingly, group 1 and 2 which both controlled the virus similarly well exhibited marked differences in the amount of total env-specific IgG. Due to the limited number of animals available for this study, these differences in the IgG titres reached significance only at week 28.
A neonatal macaque study showed that passively transferred non-nAbs did not protect the animals against oral challenge with SIVmac251 indicating that ADCC is not a main mechanism in reducing infection .
Furthermore, the data presented in the present study suggests that C activation is part of the humoral immune response. As shown by a virus capture assay, sera of the animals collected at 2 wpc induced C3-deposition on the viral surface. Although based on only four animals per group, a trend to an inverse correlation of C3-deposition on viral particles and viral load during peak viremia was observed at least within the individual groups of vaccinated monkeys (Figure 3). During the chronic phase of infection, sera of all vaccinated macaques induced C3 activation and opsonisation on SIV, independent of the viral load. C-mediated defence mechanisms have been discussed controversially in the literature. Opsonized virus particles may interact with C-receptor expressing cells, such as B-cells or dendritic cells [31–34], followed by an efficient transmission of opsonized HIV to autologous primary T-cells. At least in vitro, the infection is significantly enhanced by this mechanism. However, preliminary data indicate that in in vitro interaction assays the C-mediated increase of SIV infection is not observable in the monkey system using primary isolated macaque B- and T-cells and opsonised SIV (unpublished observation). A further mechanism of C to reduce infectivity of C-receptor-negative T-cells is the masking of viral epitopes due to the deposition of C3-fragments on the viral envelope [35, 36]. This neutralization mechanism has also been described for other viruses  and is an attractive hypothesis to explain, at least in part, the reduced viral loads observed during peak viremia.
A further result of C activation is the induction of the terminal C pathway, resulting in the destruction of pathogens. The in vitro lysis assays reduced the viral titres by a mean of 24.8% (range between 16 and 30%) when sera of immunized monkeys were tested before challenge (Table 2). Two weeks later, during peak viremia, mean lysis was 38.0% (ranging between 11 and 96%) tested in control and vaccinated monkeys. Lysis values increased further during chronic infection up to mean levels of 63.1% (range between 35 and 87%). Although C-induced lysis may contribute to the control of SIV replication, C-mediated destruction of the virus did not correlate with the control of the infection in vivo. Some animals had low peak viremia (#12127, #12142) but exhibited a poor induction of C-mediated lysis when compared to sera from other monkeys with extremely high lysis activities (#12133, #12140) but ten times higher viral loads. In line with earlier studies [11, 12, 38], no correlations between nAbs and C-mediated lysis was observed during the chronic phase of infection. Thus, Ab-mediated neutralization and C-induced lysis of retroviruses appear to represent two independent parameters which are not necessarily linked . This does not exclude the possibility that lysis may play an important role during early phases of infection before or early after seroconversion .
Beside Abs, effective SIV-specific T-cell responses are important for controlling viremia . Recently published INF-γ ELISPOT data from the present vaccination trial revealed increased cellular immune responses in cohort 2 compared to group 1 . As both groups controlled the viral loads at comparable levels, it is presently unclear to which extent the cytotoxic T-lymphocyte response is the main contributor for the reduced peak viremia and viral load reduction in the chronic phase of infection.
With this rhesus macaque study it was demonstrated that priming with SCIV [VSV-G] and boosting with both Ad5-SIV vectors or SCIV [MLV] mount humoral immune responses comparable to that of live-attenuated immunodeficiency virus vaccines [40, 41], which may contribute to the significant reduction in viral load observed in animals of group 1 and 2 after challenge. This encourages tonsillar/mucosal immunization strategies which may simplify vaccine application in the future. Thus, more efforts in research further investigating this mucosal delivery route are warranted.
Materials and methods
Young adult rhesus monkeys (Macaca mulatta) were imported from China through R.C. Hartelust BV, Tilburg, the Netherlands. Monkeys of both sexes were antibody negative for simian T-lymphotropic virus type 1, simian D-type retrovirus and SIV. Viral application, physical examinations and bleeding were done under ketamine anaesthesia. The nonhuman primate study was performed at the German Primate Centre according to paragraph 8 of the German Animal Protection law which complies with EC Directive 86/609, with project licence 509.42502/08-04.03 issued by the District Government Braunschweig, Lower Saxony.
Vaccination strategies, challenge and specimen collection
The study was conducted on 16 monkeys (Table 1). In group 1, four macaques were immunized with SCIV [VSV-G]  via tonsillar spray application at 0 and 4 wpi, as described recently [16, 43], and boosted by the same route with Ad5-SIV expressing gag-pol or env-rev at 8 and 12 wpi. Group 2 consisted of four monkeys which were immunized iv with SCIV [VSV-G] and boosted intramuscularly with Ad5-SIV 8 wpi. In group 3, four monkeys were primed with SCIV [VSV-G] iv and boosted with SCIV [MLV] iv at 8 wpi. SCIV [MLV] were prepared as described for SCIV [VSV-G] by just replacing the VSV-G expression plasmid by pHIT456 , an expression plasmid for amphotropic MLV env. Group 4 monkeys served as controls, two (#12129 and #12130) of which were immunized with an adenoviral vector containing a green fluorescent protein gene (Ad5-GFP)  via the tonsils at 8 and 12 weeks after the initiation of the experiment. The other two controls (#12134 and #12141) were immunized with Ad5-GFP intramuscularly at week 8. All macaques were challenged with approximately 2000 TCID50 of SIVmac239 [46, 47] via the tonsils 20 wpi. Sera from vaccinated and control animals were collected periodically as indicated in the figures. The heat-inactivated (hi; 56°C, 30 min) serum samples of the monkeys were used to analyze for Ab responses. As a source of complement, a pool of normal monkey serum (NMS) from untreated donors was used.
Determination of viral loads
Viral RNA in plasma was determined by quantitative real-time PCR as previously reported . In order to quantify plasma viral load, standard RNA templates were generated from the p239Sp5' plasmid (kindly provided by R. M. Ruprecht, Dana-Farber Cancer Institute, Boston, USA; ) with a detection limit of 10 viral particles per ml of plasma.
SIV p27 antigen assay
SIVmac251 replication was determined by ELISA against the p27 core protein as described recently .
SIV neutralization assays
Levels of nAbs against SIVmac251 in the sera of immunized and infected macaques were measured using a yield reduction assay . Briefly, sera diluted 1:50 were incubated with serial dilutions of SIVmac251 (25 μl serum, 25 μl virus, six replicates per dilution) in U96 microtitre plates (1 hour at 37°C). Then 150 μl of a C8166 cell suspension (2000 cells) was added. The cultures were lysed after a 7 day incubation at 37°C and virus replication in individual wells was measured by a sensitive gag-based antigen capture ELISA. Wells, giving OD values above threshold (mean of uninfected wells + 5× standard deviations), were scored positive, and the TCID50 for the virus in the presence of each serum was calculated. The yield reduction for each sample was then calculated as the virus titre in the absence of serum divided by the titre in the presence of serum.
Measurement of SIV-specific IgG
Flow cytometry was used to evaluate SIV-specific IgG responses. HSC-Fcells (provided by the EU-program EVA/MRC (QLKZ-CT-1999-00609))  were infected with SIVmac251. After washing, cells (5 × 105/analysis) were incubated on ice with hi-sera from vaccinated and infected animals (1:50, 30 minutes, two replicates per sample performed in duplicate). SIV-specific antibodies bound to infected cells were stained with a FITC-labelled anti-human IgG (Dako F0202, Glostrup, Denmark). As a negative control, hi-NMS of healthy untreated donors was used. Samples were analysed by flow cytometry using Cell Quest software (Becton Dickinson, Franklin Lakes, New Jersey, USA). Data given in the figures represent mean-fluorescence intensities (MFIs).
In vitro opsonisation and virus capture assay
Hi-monkey samples (1:50, two replicates per sample performed in double) from the vaccinated, and infected animals were incubated with SIVmac251 (160 ng/ml p27, TCID50 = 5.9 × 105log) for 30 minutes at 4°C in order to allow for the binding of the induced env-specific IgGs. Subsequently, NMS was added in a 1:10 dilution as a source of C. Hi-NMS was used as control. Samples were further incubated for 30 minutes at 37°C. To remove unbound antibodies and remaining C proteins, the virus was pelleted and re-dissolved in RPMI1640 medium. The opsonisation of the virus with C3 fragments was determined by a virus capture assay as described previously . Depending on the amount of C3 deposited on the viral surface, opsonised virus was retained in the ELISA plate. Virus was lysed by RPMI/1%Igepal and quantified by a p27-ELISA.
Continuous data are presented as means ± standard deviations, with medians in parenthesis. Kolmogorov-Smirnov-tests were conducted in order to test for Gaussian distribution of plasma and cell-associated viral load, nAbs, SIV-specific IgG titres, lysis, as well as capture parameters. Since the above variables showed significant deviation from normality at an Alpha-Level of 0.05, non-parametric tests were used throughout the analyses. We used the Kruskal-Wallis-H-Test to assess overall differences between control monkeys and immunized groups, with post-hoc Mann-Whitney-U-Tests to compare pair-wise differences between groups. Non-parametric Spearman correlation was used to investigate associations of lysis parameters. Two-sided p-values < 0.05 were considered statistically significant. All statistical analyses were conducted using SPSS 15.0 (SPSS Inc., Chicago, Illinois, USA).
antibody dependent cellular cytotoxicity
mean fluorescence intensities
murine leukemia virus
normal monkey serum
simian immunodeficiency virus
median tissue culture 50% infectious dose
G protein of vesicular stomatits virus
weeks post challenge
weeks post immunization
The authors are supported by the 6th frame work of the EU (QLK-CT-2002-00882, TIP-Vac 012116), grants of the Austrian Research Fund FWF (P17914 to HS), the Ludwig Boltzmann Institute of AIDS Research and the Federal Government of Tyrol. Different cell lines and reagents were obtained from the Centralized Facility for AIDS Reagents, NBSC, UK (EU-program EVA/MRC (QLKZ-CT-1999-00609)). The secretarial support of L. Hahn is gratefully acknowledged.
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