EFFECTIVE, LOW TITER, ANTIBODY PROTECTION AGAINST LOW-DOSE REPEATED MUCOSAL SHIV CHALLENGE IN MACAQUES

Abstract

Neutralizing antibodies are thought crucial to HIV vaccine protection but a major hurdle is the high antibody concentrations likely required as suggested by studies in animal models¹. However, these studies typically apply a large virus inoculum to ensure infection in control animals in single challenge experiments. In contrast, most human infection via sexual encounter probably involves repeated exposures to much lower doses of virus^2–4. Therefore, animal studies may have overestimated protective antibody levels in humans. To investigate the impact of virus challenge dose on antibody protection, we repeatedly exposed macaques intravaginally to low doses of a CCR5 coreceptor-using SHIV (an HIV/SIV chimera) in the presence of antibody at plasma concentrations leading to relatively modest neutralization titers of the order of 1:5 IC₉₀ values in a PBMC assay. An effector function deficient variant of the neutralizing antibody was also included. The results show that a significantly greater number of challenges are required to infect animals treated with neutralizing antibody than control antibody-treated animals, and the notion that effector function may contribute to antibody protection is supported. Overall, the results imply that lower levels of antibody than considered hereto may provide benefit in the context of typical human exposure to HIV-1.

Much of what we know about antibody protection against HIV comes from studies using passively administered broadly neutralizing human monoclonal antibodies or monospecific neutralizing polyclonal antibodies in animal challenge models^5–11, including intravenous (i.v.), vaginal and rectal challenge in macaques. The hallmark of most of these studies is that protection, in the form of sterilizing immunity, is achieved at relatively high serum neutralization titers corresponding to high antibody concentrations. The most quantitative of these studies suggest that sterilizing immunity requires serum antibody concentrations at least two orders of magnitude greater than in vitro neutralizing concentrations^10,11. However, this estimate is quite approximate and dependent upon, among other parameters, the neutralization assay used. Even so, the data have convinced many researchers that achieving sterilizing immunity via antibodies alone is extremely challenging and a more realistic goal for vaccine-induced antibodies has been viewed as blunting infection and relying on vaccine-induced cellular immunity to clear, or, failing that, control infection. However, as noted above, a limitation of macaque protection studies is the use of high viral challenge doses to ensure all control animals become infected with a single challenge. Yet, it is well established that the average probabilities for heterosexual transmission in human exposures are low and dependent upon the viral burden in the donor and susceptibility factors associated with the donor and recipient such as the presence of sexually transmitted diseases (STDs). Transmission frequencies on the order of 1:1,000 per coital act have been reported in chronic infection of the donor^2–4,12, increasing by about an order of magnitude in acute infection^2,3,12,13. The amount of virus, albeit estimated by quantitative PCR rather than infectivity, contained in a typical macaque challenge is much higher than would be found, for example, in the semen ejaculate of an acutely infected man^12–15. Indeed, viral inoculums typically average 5 × 10⁵ copies per ejaculate, with a reported maximum of about 2 × 10⁷ copies¹², whereas we found that a high-dose 300 TCID₅₀ (50% tissue culture infectious doses) inoculum of SHIV_SF162P3 contains about 10⁸ viral copies.

In order to investigate antibody protection against viral challenge doses that may better represent those encountered in human heterosexual exposure, we utilized a low-dose repeated mucosal challenge model^14,16 in which a reduced virus dose requires several challenges to infect untreated animals, but yet eventually infects all animals with a reasonable number of challenges. In this model, we could expect to observe benefit provided by antibody if the number of challenges required for infection in treated animals was greater than the number of challenges required for infection in controls.

The human monoclonal antibody b12 neutralizes a broad range of HIV isolates from a variety of clades^17,18 through recognition of a conserved epitope overlapping the CD4-binding site of gp120¹⁹. A high serum concentration of b12, corresponding to about 75-fold the IC₉₀ in a PBMC assay and 3,000-fold the IC₅₀ in a pseudotyped virus assay provided 90% protection against a high-dose vaginal challenge with SHIV_SF162P3²⁰. In addition in that study, the importance for protection of the interaction of b12 with Fc receptors was established by comparison of b12 and engineered b12 variants²⁰.

Here, we explored the question of whether a relatively low b12 neutralizing antibody titer could provide benefit to macaques in the low-dose repeated challenge model and simultaneously compared protection by the effector function-deficient b12 variant LALA. Based on earlier studies ^14,16,21, we began the experiment with repeated 3 TCID₅₀ SHIV_162P3 vaginal challenge. With only a single animal infected after 11 challenges, we increased the viral dose to 10 TCID_50. This dose corresponds to approximately 2.65 × 10⁶ viral RNA (vRNA) copies, an amount somewhat higher than typically found in human semen during acute infection^12,13 but substantially lower than traditional high-dose challenges with SHIV_SF162P314.

The study involved a total of 14 animals, consisting of 4 isotype control-treated animals, 5 animals receiving wild-type b12, and 5 animals receiving the LALA variant, which has similar neutralizing activity as b12 but does not mediate Fc effector functions ²⁰. Animals were i.v.-treated weekly (Thursday) with 1 mg/kg of antibody to maintain serum levels, based on previously reported half-lives²⁰. This dose of b12 antibody is far less than the 25 mg/kg dose that provides 90% protection against high-dose challenge with SHIV_SF162P3 ²⁰ and provides negligible protection against high-dose challenge with SHIV_SF162P410. Intravaginal challenges were administered twice weekly (Friday and Monday) and blood drawn regularly to monitor viral infection, passively transferred antibody levels and serum neutralizing activity. Supplementary Figure 1 details the entire treatment course for each animal and Supplementary Table 1 summarizes antibody treatments, viral challenges, detection and day-of-peak viremia in plasma.

As shown in Figure 1 notably more challenges were required to infect b12-treated than control animals and also suggests that somewhat fewer challenges may be required to infect LALA variant-treated than wild-type b12-treated animals. One animal (b12-treated, BF68) remained uninfected after 40 consecutive 10 TCID₅₀ challenges. We investigated the magnitude of protection using three approaches. First, we used an adapted Kaplan-Meier analysis (Fig. 2) in which the percent of animals remaining uninfected is plotted against the number of 10 TCID₅₀ viral challenges. To prevent positive bias, we also included the animal BK10 that was infected in the 3 TCID₅₀ challenge series as if it was infected by the first 10 TCID₅₀ challenge (see above and Suppl. Fig. 2). The three survival curves are significantly different (p=0.0377). A comparison of the individual pairs of Kaplan-Meier curves reveals that LALA is significantly different from control (p=0.0027) while a (borderline) non-significant difference for b12 versus control (p=0.056) is seen due to the strong penalty incurred by including BK10 in the analysis. The same analysis excluding BK10 would indicate a significant difference (p=0.0058). The LALA and b12 groups did not differ significantly from each other. Second, we calculated hazard ratios for b12 and LALA-treated animals with a Cox-proportional hazard model that estimates the relative risk of infection for each of the treatment groups versus controls. Treatment with b12 was found to reduce the infection risk by about 20-fold. The risk reduction for LALA treatment was approximately 10-fold (Table 2a). Third, we calculated the reduction in infection susceptibility as described by Regoes, et al ²² by tallying the total number of 10 TCID₅₀ virus challenges required to infect all animals within each group (within the limits of the experiment). As shown in Table 2b b12-treated animals (p=0.0016) as well as LALA-treated animals (p=0.0145) only became infected after a significantly larger number of challenges compared to the control group. It should be noted that this number is underestimated for b12 in this type of analysis, as one b12-treated animal remained uninfected at the end of the experiment. Overall, our analyses suggest that there is a significant difference in the protection afforded by the repeated administration of 1 mg/kg of both b12 antibody and LALA variant as compared to treatment with the isotype control antibody. The approximately two-fold difference in b12 and LALA hazard ratios and the observation that b12-treated animals resisted nearly twice as many challenges as LALA-treated animals (104 versus 61) reflects the trend previously described in a high-dose virus challenge for the effector function-crippled LALA variant to be less effective in protection than the fully effector function-competent wild type b12 antibody²⁰. An analysis of peak viremias suggests a trend towards lower peak viremias in the b12-treated group compared to controls although this difference does not achieve significance (Suppl. Fig. 3). However, there is a significant difference (p = 0.016), about 2 orders of magnitude, between peak viremias in the b12 and LALA–treated animals, again consistent with an impact of effector function on anti-viral activity.

Figure 1. Protection by b12 and variant LALA during vaginal low-dose repeated challenge with SHIV_SF162P3.

Female Indian rhesus macaques were treated weekly with 1 mg kg⁻¹ of either b12 or b12 effector function variant LALA or an isotype control antibody (anti-dengue, DEN3) and challenged vaginally twice weekly. The viral challenge dose began at 3 TCID₅₀ and was subsequently increased to 10 TCID₅₀ SHIV_SF162P3. a, All animals in the isotype control groups became virus positive after a maximum of 4 challenges of 10 TCID₅₀. 4 out of 4 animals were infected after a total number of 10 challenges of 10 TCID₅₀. b, 3 b12-treated animals were virus positive after 6, 23, and 38 viral challenges of 10 TCID₅₀, respectively, and 1 animal (BF68) remained virus negative after 40 challenges. 3 out of 4 animals were infected after a total number of 107 challenges of 10 TCID₅₀. BK10 was infected after 6 challenges of 3 TCID ₅₀. (See Suppl. Fig. 2.) c, Plasma virus was observed in the LALA-treated animals following 6, 8, 12, 17, and 23 viral challenges, respectively. 5 out of 5 animals were infected after a total of 66 challenges of 10 TCID₅₀. Viral challenges and i.v. antibody treatments were suspended after positive detection of virus in plasma but the course of infection was monitored for several weeks. The SIV viral RNA (vRNA) assay detection limit is 125 copies ml^–1 (log 2.1).

Figure 2. Kaplan-Meier analysis and magnitude of protection in low-dose (10 TCID₅₀) repeated challenge by b12 and LALA treatment.

a. Kaplan-Meier analysis. The percent of animals remaining uninfected is plotted against the number of 10 TCID₅₀ viral challenges (compare Fig. 1). A single animal (BK10; b12-treated) became infected during the initial repeat 3 TCID₅₀ challenge (see Suppl. Fig. 2). To allow inclusion of this animal in the analysis, it is included as if it was infected in the first 10 TCID₅₀ challenge. The Kaplan-Meier survival curves are significantly different from each other (p = 0.0377; Log-rank (Mantel-Cox) test)

b. The reduction in infection susceptibility by b12 and LALA treatment is estimated by counting the number of challenges that did or did not result in infection. Again the animal BK10 is included in this analysis as being infected in the first 10 TCID₅₀ challenge. Both b12 (p=0.0016) and LALA (p=0.0145) are significantly different from the control (Fisher’s exact test).

Table 2. Statistical analyses comparing relative risk of infection between treatment groups.

(a) The hazard ratios for b12 and LALA-treated animals are calculated using a Cox-proportional hazard model. It shows that b12 and LALA treatment significantly reduced the risk of infection at each challenge by a factor of 21 and 10 times, respectively. (b) The reduction in infection susceptibility ²² is also demonstrated by comparing the total number of challenges resulting in infection to the total number of challenges not leading to infection.

a Cox-proportional hazard model
Group	Hazard ratio	95% CI of ratio
b12 vs control	21.3*	1.7; 260.9
LALA vs control	10.1	1.0; 101.0

b Infection susceptibility
Group	Number of 10 TCID50 challenges leading to infection	Number of 10 TCID50 challenges not leading to infection
Control	4	6
b12	4*	104
LALA	5	61

^*To prevent positive bias, BK10 has been included in this analysis as if it was infected in first challenge.

We determined antibody serum concentrations throughout the course of the experiment by ELISA (Supplementary Fig. 4). Considerable variations in individual serum concentration were found, but no significant correlation was found between average concentration and the number of challenges to infection. Likewise, the appearance of infection did not correlate with the magnitude of the antibody concentration at the estimated time of infection (10 – 17 days prior to detection of virus). Neutralizing antibody titers in sera were assessed in a pseudovirus assay and were as expected based on previous studies^10,20 given the antibody concentrations measured by ELISA (Supplementary Table 2). Average b12 concentrations for challenges not resulting in infection was relatively low, about 40 μg/ml, corresponding to an average 1:200 IC₅₀ titer in a pseudovirus assay and to an estimated 1:5 IC₉₀ titer in a PBMC assay (Table 1). MHC genotyping revealed that there was no apparent correlation with the allelic profiles of the animals in this study that would account for any unusual ability to resist infection (Supplementary Table 3).

Table 1.

Average serum antibody concentrations and neutralization titers in macaques repeatedly challenged with a low dose of SHIV_162P3 in the period before they became infected.

Animal	# of i.v. Ab treatments without infection	Average serum Ab [μg/ml]1	Average IC50 pseudovirus assay2	Average IC90 PBMC assay3
LALA-treated
DC60	9	25	1:125	1:3
BG27	11	26	1:130	1:3
BL45	13	46	1:230	1:6
CL95	15	33	1:165	1:4
BA16	18	37	1:185	1:5
b12-treated
BK10	5	31	1:155	1:4
BR05	9	25	1:125	1:3
BF53	19	60	1:300	1:8
N364	27	53	1:265	1:7
BF684	28	40	1:200	1:5

¹Average serum concentration of transferred b12 and LALA for each macaque prior to infection.

²Average neutralization titer estimated from the average serum concentrations and b12 and LALA IC₅₀ values in the pseudovirus assay (=0.2 μg/ml).

³Average neutralization titer estimated from the average serum antibody concentration and b12 and LALA IC₉₀ values in a PBMC-based assay (=8 μg/ml).

⁴BF68 did not become infected after 40 challenges at 10 TCID₅₀.

In summary, we have shown that neutralizing antibody can provide clear benefit against repeated low-dose SHIV challenge in the macaque model at low serum antibody concentrations corresponding to modest neutralization titers. There is a concern that low-dose challenge models may be “lowering the bar” too much in terms of the requirements for protection. In this context, we note that oral chemoprophylaxis is possibly less, and certainly not more, protective against SHIV_SF162P3 challenge in the low-dose repeated challenge model, arguing that the model is not intrinsically and universally more susceptible to protective intervention²¹. If translated into protection against HIV infection in humans, the findings are a promising development for HIV vaccine design. Serum neutralizing antibody titers in the approximate range of 1:200 (IC₅₀ values in a pseudovirus assay) corresponding to about 1:5 (IC₉₀ values in a PBMC assay) increased the number of low-dose challenges to achieve infection here by at least an order of magnitude. If vaccination in humans led to a similar decrease of transmission rate, then one might expect a significant impact on the pandemic. Neutralizing titers above are near or below those described in the sera of a significant proportion of HIV-infected donors against multiple isolates from different clades^23–27 suggesting that such titers may be achieved with appropriate immunogens. Finally, the data further support the contribution of effector function in antibody resistance to HIV infection, underscoring the notion that the ability of an immunogen to elicit extra-neutralizing antibody activities in addition to neutralization should be assessed in vaccine evaluation.

METHODS

Macaques

All protocols for female Indian rhesus macaques were reviewed and approved by the Institutional Animal Care and Use Committees. The animals were housed in accordance with the American Association for Accreditation of Laboratory Animal Care Standards. At the start of all experiments, all animals were experimentally naïve and were negative for antibodies against HIV-1, SIV, and type D retrovirus. Virus challenge and i.v. antibody protocols are more fully described elsewhere¹⁰.

Challenge virus

The virus used in this study was SHIV_SF162P passage 3, which has been described elsewhere^28,29. SHIV_SF162P3 retains the R5 phenotype of HIV-1_SF162. SHIV_SF162P3.propagated in phytohemagglutin (PHA)-activated rhesus macaque peripheral blood mononuclear cells (PBMC), was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (Cat. No. 6526; Contributors: Drs. Janet Harouse, Cecilia Cheng-Mayer, and Ranajit Pal).

b12 and variant antibody LALA

IgG1 b12 is a human antibody (IgG1, κ) that recognizes an epitope overlapping the CD4 binding site of gp120^17,19. Variants of b12 were created by site-directed mutagenesis as previously described³⁰.

Antibody production

Recombinant IgG1 (wild type b12, isotype control, and b12 LALA variant (L234A, L235A) were expressed in Chinese hamster ovary (CHO-K1) cells in glutamine-free custom formulated Glasgow minimum essential medium (GMEM Selection Media) (MediaTech Cellgro) ¹⁰. The isotype control antibody DEN3, an anti-Dengue NS1 human IgG1 antibody, was used in this study. For large-scale tissue culture, media was supplemented with 3.5% Ultra Low Bovine IgG Fetal Bovine Serum (Invitrogen) and grown in 10-layer Cellstacks and Cell Cubes (Corning). Antibodies were purified using Protein A affinity matrix (GE Healthcare), and dialyzed against phosphate-buffered saline (PBS). Care was taken to minimize endotoxin contamination, which was monitored using a quantitative chromagenic Limulus Amoebecyte Lysate assay (Cambrex) performed according to the manufacturer’s recommendations. Antibody used for the passive transfer experiments contained <1 IU of endotoxin mg^–1.

Plasma viral loads

The quantity of SIV viral RNA genomic copy equivalents (vRNA copy Eq/ml) in EDTA-anti-coagulated plasma was determined using a quantitative reverse-transcription PCR (QRT-PCR) assay as previously described³². Briefly, vRNA was isolated from plasma using a GuSCN-based procedure as described³¹. QRT-PCR was performed using the SuperScript III Platinum® One-Step Quantitative RT-PR System (Invitrogen, Carlsbad, CA). Reaction mixes did not contain bovine serum albumin (BSA). Reactions were run on a Roche LightCycler 2.0 instrument and software. vRNA copy number was determined using LightCycler 4.0 software (Roche Molecular Diagnostics, Indianapolis, IN) to interpolate sample crossing points onto an internal standard curve prepared from 10-fold serial dilutions of a synthetic RNA transcript representing a conserved region of SIV gag.

ELISA

b12 and variant antibody concentrations in macaque sera were determined by ELISA against recombinant monomeric HIV-1 gp120_JR-FL (kindly provided by Progenics) and is fully described elsewhere¹⁰.

Neutralization assays

Neutralization titers in animal sera were reported by Monogram Biosciences after preparation of an HIV-1 envelope pseudotyped luciferase SHIV_SF162P3 capable of single-round replication and performed as previously described³⁵.

MHC genotyping

MHC genotyping by sequence-specific PCR was performed by the University of Wisconsin Genotyping Core with support of NIH grant 5R24RR16038-6 awarded to David I. Watkins and previously described ³⁴.

Statistics

The isotype control groups consisted of a total of 4 animals (n=4), and each of the treated groups consisted of 5 animals (n=5). Statistical analyses were performed using Graph Pad Prism for Mac Software, version 5.0a (Graph Pad Software Inc., San Diego, CA, 2005). A Kaplan-Meier Survival Analysis was performed for Figure 2. The alpha level was 0.05.