Evaluation of Passively Transferred, Nonneutralizing Antibody-Dependent Cellular Cytotoxicity-Mediating IgG in Protection of Neonatal Rhesus Macaques against Oral SIVmac251 Challenge

Ruth H. Florese, Koen K. A. Van Rompay, Kris Aldrich, Donald N. Forthal, Gary Landucci, Madhumita Mahalanabis, Nancy Haigwood, David Venzon, Vaniambadi S. Kalyanaraman, Marta L. Marthas and Marjorie Robert-Guroff
J Immunol September 15, 2006, 177 (6) 4028-4036; DOI: https://doi.org/10.4049/jimmunol.177.6.4028

Abstract

Previously, Ab-dependent cellular cytotoxicity (ADCC) was significantly correlated with reduced acute viremia upon intrarectal SIVmac251 challenge of immunized rhesus macaques. To directly assess ADCC protective efficacy, six neonatal macaques were infused s.c. with immune IgG (220 mg/kg) purified from the immunized animals and positive for ADCC and Ab-dependent cell-mediated viral inhibition (ADCVI) activities. Six neonates received control IgG. The neonates were challenged twice orally with 105 50% inhibiting tissue culture-infective dose of SIVmac251 2 days post-IgG infusion. At challenge, plasma of neonates that received immune IgG did not neutralize SIVmac251 but had geometric mean ADCC titers of 48,130 and 232,850 against SIVmac251-infected and gp120-coated targets, respectively. Peak ADCVI activity varied from 62 to 81%. ADCC activity declined with the 2-wk IgG half-life but was boosted at wk 4, together with de novo ADCC-mediating Abs in controls, by postchallenge viremia. ADCVI activity was similarly induced. No protection, assessed by viral burdens, CD4 counts, and time to euthanasia was observed. Possible factors contributing to the discrepancy between the previous correlation and lack of protection here include: the high oral challenge dose compared with the 400-fold lower intrarectal dose; the challenge route with regard to viral dissemination and distribution of infused IgG; insufficient NK effector activity and/or poor functionality in newborns; insufficient immune IgG; and the possibility that the previous correlation of ADCC with protection was augmented by cellular immune responses also present at challenge. Future studies should explore additional challenge routes in juvenile macaques using higher amounts of potent IgG preparations.

Antibody-dependent cell-mediated cytotoxicity (ADCC)3 is a powerful immune mechanism that can eliminate virus-infected cells via effector cells armed with viral-specific Abs. The ADCC process requires three components: target cells expressing a surface Ag, Ag-specific Abs, mainly of the IgG isotype, and FcγR-bearing effector cells such as NK cells, γδT cells, neutrophils, and macrophages. Abs binding to both target Ags and effector cell FcγRs induce target killing by lysis and/or apoptosis.

Abs mediating ADCC are among the first antiviral immune responses to occur following infection. In HIV-infected individuals, Abs with ADCC activity appear early in acute infection, often preceding a neutralizing Ab response (1, 2). In HIV-infected patients, as in SIV-infected rhesus macaques, ADCC responses have been associated with a better clinical outcome and/or lower viral loads (3, 4, 5, 6). Yet to date, the contribution of ADCC activity to protection against HIV or SIV infection has not been fully elucidated.

Ab-dependent cell-mediated virus inhibition (ADCVI) is closely related to ADCC in that it relies on interactions between an infected target cell, Ab, and FcR-bearing effector cells. However, rather than target cell lysis, ADCVI measures virus inhibition from infected target cells and may involve both cytotoxic and noncytotoxic mechanisms. ADCVI has been associated with the fall in viremia during acute HIV infection and may underlie the protective effect of passively infused, nonneutralizing Ab (7, 8).

Previously, we reported strong protection against intrarectal SIVmac251 challenge following two mucosal immunizations with one or more replication-competent Ad type 5 host range mutant (Ad5hr) expressing SIVenv/rev, SIVgag, and/or SIVnef and boosting with SIV gp120 protein or a polypeptide “peptomer” representing the CD4 binding site of the SIV envelope (9). High-titer SIV envelope-specific binding Abs were induced by the vaccine regimen and shown to correlate with reduced acute phase viremia. Notably, these binding Abs did not neutralize primary SIVmac251. Both serum Abs and purified IgG from the immunized rhesus macaques mediated ADCC activity that was correlated with reduced acute viremia (6). These results implied that the Abs possessed a functional activity different from virus neutralization that contributed to viremia control.

Classically, the role of Abs in protecting against infection or disease has been studied by passive transfer experiments, whereby serum, immune globulin, or Ab preparations are infused into a host before or shortly after challenge with an infectious agent. Numerous such studies have shown protective efficacy of HIV or SIV Abs against i.v., vaginal, or oral challenges in nonhuman primates. Both neutralizing mAbs specific for the HIV envelope (10, 11, 12) and polyclonal immune globulin with neutralizing activity against HIV or SIV (13, 14) have been shown to be protective. In contrast, the contribution of nonneutralizing Abs to protection has been rarely assessed. Passive transfer of SIV hyperimmune serum lacking the ability to neutralize the SIVmac251 challenge virus protected newborn rhesus macaques against oral SIV challenge (15). In vitro studies recently attributed this protection to ADCVI (7). No passive transfer studies have directly assessed ADCC activity in the absence of other functional activities in SIV or simian HIV systems.

To elucidate the role of the previously described vaccine-elicited ADCC-mediating Abs in protection against SIVmac251 (6, 9), we purified IgG from a pool of prechallenge macaque sera and plasma and conducted a passive transfer experiment. We also evaluated the immune IgG transferred and macaque plasma pre- and post-SIVmac251 challenge for ADCVI activity. As limited IgG was available, we chose the neonatal SIV rhesus macaque model. Oral inoculation of newborn macaques with pathogenic SIV is a useful model of human infant HIV infection (16) and has been used in evaluating both immunologic and therapeutic intervention strategies (15, 17, 18). The small size of the newborns allowed us to include sufficient animals in the study to provide statistical power. In the present study, we report the challenge outcome and results of extensive immunologic analyses pre- and postchallenge.

Materials and Methods

Preparation of IgG fractions

Sera and plasma previously collected from 31 vaccinated rhesus macaques and stored at −70°C were thawed and combined as pool A. The macaques had been immunized intranasally and then intratracheally with an Ad5hr expressing SIVenv/rev, with or without additional Ad5hr encoding SIVgag and/or SIVnef (9). The animals were subsequently boosted twice i.m. with native SIV gp120 protein in MPL-SE adjuvant. Samples obtained 2 wk after the second protein booster immunization (wk 38 postinitial immunization) and at the time of challenge (wk 42) were pooled. The time-of-challenge samples were previously shown to possess high-titered Abs that mediated ADCC activity (6). Sera and plasma from mock-immunized control macaques from the same study (after monophosphoryl lipid A-stable emulsion immunizations, wk 26, 30, 34, 38, and 42) were also thawed and combined as pool B. To obtain sufficient control sera and plasma, samples obtained following the last mock-booster immunization (wk 38 and 42) were added from a previous study in which macaques received two intranasal administrations of empty Ad5hr vector and two immunizations with QS-21 adjuvant only (19). The final volume of pool A was 135 ml, whereas pool B contained 109 ml.

IgG was purified from pools A and B by the Immunology Core, Humoral Immunity Subcore of the University of Washington Center for AIDS Research, Seattle Biomedical Research Institute. The purification method was described previously (14). Briefly, pools A and B were thawed, heat inactivated at 56°C for 45 min, clarified by centrifugation, and filtered through a 0.2-μM mini capsule filter before purification on protein G-Sepharose (Amersham Biosciences) equilibrated with PBS. Pool B was purified first, and then pool A was purified using the same protein G column. IgG was eluted with 0.5 M acetic acid (pH 3.0) and neutralized with 3 M Tris (pH 9.0). IgG containing fractions, identified by OD (OD280), were verified by electrophoresis on reducing SDS-polyacrylamide gels. Peak fractions were pooled, concentrated, dialyzed extensively in PBS, and filtered through a 0.2-μM filter. The IgG preparations contained <5 endotoxin U/ml. Coomassie-stained gels indicated ∼95% purity for both pool A and B IgG preparations. Pool A contained 1118 mg of immune IgG (13 mg/ml) and pool B contained 739 mg of control IgG (11.2 mg/ml).

Animals and assessment of MHC class

Newborn rhesus macaques (Macaca mulatta) were negative for SIV, HIV type 2, type D retrovirus, and simian T cell lymphotropic virus type 1. They were hand-reared at the California National Primate Research Center and housed following the guidelines of the American Association for Accreditation of Laboratory Animal Care. Strict adherence to the “Guide for Care and Use of Laboratory Animals” (20) was observed. When necessary, animals were immobilized with 10 mg/kg ketamine hydrochloride (Parke-Davis) injected i.m. Six animals were randomly assigned at birth to the experimental group and received immune IgG from pool A. Six formed the control group and received control IgG from pool B. MHC typing was performed by the Rhesus Macaque MHC Typing Core, University of Wisconsin Hospital and Clinics. Two neonates in the experimental group (36460 and 36495) and one in the control group (36475) were positive for the Mamu-A*01 allele. Weights of the neonates ranged from 0.45 to 0.61 kg with a mean of 0.5 kg for both groups.

IgG administration and SIVmac251 challenge

Within 4 days of birth, neonates were s.c. administered either 220 mg/kg immune or control IgG while under ketamine anesthesia. Two days later, the animals were bled and orally administered two sequential high-dose oral challenges, 24 h apart, with 105 50% inhibiting tissue culture-infective dose (TCID50) of SIVmac251 (lot no. 2/02 propagated on rhesus PBMC; titer of 8.6×108 viral RNA copies/ml). Animals were bled on day 7 postchallenge and then at weekly or bimonthly intervals for routine monitoring of blood counts and lymphocyte subsets, viral loads, and Ab responses. Plasma samples were stored at −70°C until use. The macaques were also monitored for weight, general health, and clinical signs and symptoms of disease progression. Animals were euthanized when necessary according to previously defined criteria (21).

Postchallenge virologic monitoring and lymphocyte phenotyping

SIV RNA in plasma was quantified by a bDNA signal amplification assay, specific for the SIVmac251 pol gene (22). Lymphocyte phenotypic analysis was performed using three- and four-color flow cytometry as described previously (22).

Ab assays

The ability of Abs to mediate ADCC activity was assessed using the rapid fluorometric ADCC (RFADCC) assay described elsewhere (23). Briefly, CEM-NKr cells (AIDS Research and Reference Reagent Program, National Institutes of Allergy and Infectious Diseases) coated with SIVgp120 or H9 cells chronically infected with SIVmac251 were used as targets. The target cells were dual labeled with the membrane dye, PKH-26 (Sigma-Aldrich), and CFSE (Molecular Probes), a vital dye that is rapidly lost when cell membranes are damaged. Labeled targets were resuspended in RPMI 1640 medium containing 10% FCS (R-10) and allowed to react with heat-inactivated (56°C, 30 min) serially diluted plasma in a 96-well microtiter plate for 30 min at room temperature. Human PBMC used as effector cells were added at a 50:1 E:T ratio. The reaction mixture was incubated for 4 h at 37°C in 5% CO2, after which the cells were fixed with 3.7% paraformaldehyde for flow cytometry. Controls included nonstained and single-stained target cells. Nongated events (50,000) in duplicate wells were acquired within 18 h using a FACSCalibur instrument (BD Biosciences). Acquisition was done using CellQuest software, and data analysis was performed with WinMDI 2.0. Percent ADCC cell killing is reported as the percentage of membrane-labeled target cells having lost the viability dye, i.e., percentage of CFSEnegative within the PKH-26high gate. ADCC titers are defined as the reciprocal dilution or IgG concentration at which the percent ADCC killing was greater than the mean percent killing of the negative control plus 3 SDs.

The ADCVI assay was based on methods previously described for measles virus and HIV (8, 24). CEMx174 target cells were infected with the SIVmac251 challenge stock at a multiplicity of infection of 0.01. After adsorption for 1 h, cells were washed, incubated in 5% CO2 at 37°C for 48 h in medium, and washed again. Infected target cells (5 × 104) were next plated in 96-well round-bottom microtiter plates, and various dilutions of plasma were added along with human PBMC effector cells at an E:T ratio of 10:1. Plasma in the absence of effector cells was also tested. After 7 days incubation at 37°C in 5% CO2, supernatant fluid was collected and assayed for p27 by ELISA (Zeptometrix). Virus inhibition due to ADCVI was calculated as follows: percent inhibition = 100 × (1 − ([p27p]/p27n])), where [p27p] and [p27n] are the concentrations of p27 in supernatant fluid from wells containing a source of SIV-positive or -negative Ab, respectively. Mean values from experiments using two different donor cells are reported. Titer is expressed as the reciprocal of the plasma dilution at which 60% inhibition was observed.

Binding Abs to SIVmac251 gp120 were determined by ELISA (25). The binding titer was defined as the reciprocal of the plasma dilution or the IgG concentration at which the absorbance of the test plasma or IgG was twice that of a negative control serum diluted 1/200. Binding titers to whole SIV were determined as described previously (26).

Neutralizing Abs in the IgG fractions and neonate plasma against the SIVmac251 challenge stock were evaluated in sMAGI cells (27) as described previously (28). Positive controls included plasma from a macaque infected with SIVmneE11S, known to neutralize SIVmac251. End point titers of 75 and 90% are reported.

Statistical analyses

The Wilcoxon rank-sum test was used to compare viral loads and survival times between experimental and control groups and to analyze differences between postchallenge ADCVI activity in experimental and control macaques. Spearman rank correlation coefficients were calculated in analyzing ADCC titers, percent killing, and survival times.

Results

Characterization of passively transferred Ab

We previously reported that sera from vaccinated macaques at the time of challenge possessed high-titered binding Abs able to mediate ADCC activity against SIV-infected target cells (6) but lacked neutralizing activity against primary SIVmac251 (9). In the present study, we combined sera and plasma obtained at challenge (wk 42 postimmunization) and 2 wk following the last protein booster immunization (wk 38) from the same macaques to obtain an experimental pool (pool A) from which immune IgG could be purified. Sera and plasma from mock-immunized macaques were similarly pooled for preparation of control IgG (pool B). Before purification, the properties of pools A and B were examined. Serum/plasma pool A exhibited high-titered binding Ab to SIVmac251 gp120, whereas pool B was Ab negative (Table I⇓). Similarly, Abs in pool A mediated potent ADCC activity against both SIV-infected target cells and target cells coated with SIV gp120, whereas pool B was negative with either SIV-infected or gp120-coated targets.

View this table:
Table I.

Properties of sera/plasma and IgG pools used for passive transfera

Following IgG purification, the pools were reassessed for the same properties. Immune IgG from pool A exhibited a potent binding Ab titer against SIV gp120 together with ADCC titers against SIV-infected and gp120-coated target cells in the nanogram range (Table I⇑). In contrast to results with serum/plasma pool A, the immune IgG of pool A exhibited some loss of ADCC titer against gp120-coated target cells relative to the ADCC titer against SIV-infected targets, suggesting non-IgG Abs removed from the IgG pool may have possessed some ADCC activity. Pool A immune IgG was also positive for ADCVI activity, exhibiting >90% inhibition of SIVmac251 infection at the concentration tested. The control IgG of pool B lacked binding, ADCC, and ADCVI Ab activity. As expected, based on earlier results showing no neutralization of primary SIVmac251 by sera at challenge, 90% neutralization of the SIVmac251 challenge stock required a high concentration of pool A immune IgG. Pool B was negative.

Prechallenge Ab characterization

Following s.c. administration of immune or control IgG to the neonates, blood samples were obtained 2 days later, and the macaques were orally challenged twice, 24 h apart, with SIVmac251. Plasma samples obtained at challenge were evaluated for binding and functionally active Abs. The passive transfer resulted in the appearance of anti-SIV Abs in the blood of the neonatal macaques 2 days later (Table II⇓). Therefore, binding and functionally active Abs were present at the time of SIVmac251 challenge. As expected based on the similar size of the macaques, all six that received immune IgG of pool A exhibited similar binding Ab titers to whole SIV, as well as to purified native SIV gp120. Similar ADCC titers among the six neonates were also observed using both gp120-coated (geometric mean titer of 232,850) and SIV-infected targets (geometric mean titer of 48,130). No ADCC activity was observed in plasma of the control neonates. Macaques that received immune IgG also exhibited ADCVI activity, with peak inhibition ranging from 62 to 81%. Peak values were sometimes observed at plasma dilutions of 1/100 rather than the lesser 1/25 dilution due to a prozone effect (29). Plasma from only two animals exhibited >60% ADCVI activity at dilutions > 1/100. Ninety percent of neutralization titers against the challenge stock of SIVmac251 were <25 for all macaques (data not shown). Plasma samples from macaques that received control IgG were negative for binding, ADCC-mediating, and neutralizing Abs and exhibited negligible inhibition in the ADCVI assay.

View this table:
Table II.

Ab activities in the plasma of neonatal macaques at the time of challenge, following passive transfer

Results of oral SIVmac251 challenge

Following the oral SIVmac251 challenges on 2 successive days, both experimental and control macaques became infected, exhibiting high viral loads (Fig. 1⇓, A and B). No difference was observed between groups. High viremia was generally maintained over time among the neonates until they had to be euthanized due to progression to AIDS. Rhesus neonates have high CD4+ T cell counts compared with adults (21, 30, 31). Thus, this phenotype partly explains the high viral burdens because more target cells are available for viral infection. In this regard, CD4 T cell counts declined somewhat by wk 2 postchallenge but not to exceedingly low levels (Fig. 1⇓, C and D), which is consistent with previous studies on SIVmac251-infected infant macaques (21, 31). No difference in CD4 count was observed between neonates that received immune IgG or control IgG.

FIGURE 1.
FIGURE 1.

Viral loads (A and B) and CD4+ lymphocyte levels (C and D) among neonates following oral SIVmac251 challenge. Recipients of immune IgG (A and C); control IgG (B and D). An asterisk denotes Mamu-A*01-positive macaques. Values for wk 9, 10, and 11 are combined and plotted as wk 10.

We also analyzed length of survival of infant macaques in both groups. The majority of infants developed clinical AIDS and had to be euthanized between 9 and 14 wk, although one Mamu-A*01 positive neonate (36460) that received immune IgG survived for 24 wk (data not shown). Two other Mamu-A*01-positive macaques (36475 and 36495 that received control and immune IgG, respectively) were among the last macaques euthanized at wk 13 and 14, respectively. Mamu-A*01-positive macaques control viremia more effectively than Mamu-A*01-negative animals (32), and in the present study, the three Mamu-A*01-positive animals had significantly longer survival than the other nine (p = 0.027 by the exact log-rank test). The viral loads of these three macaques were also significantly lower than the other nine over wk 4, 6, 8, and 9/10 postchallenge (p < 0.02 for each by the Wilcoxon rank-sum test). However, after taking the Mamu-A*01 factor into account, there was still no significant difference in time to death or viral burden between the two groups of neonates.

Postchallenge Ab activity

To better understand the similar challenge outcome between the two groups of macaques, we evaluated the spectrum of Ab activities postchallenge. Initially, we assayed binding Abs specific for SIVgp120 over the course of infection. The gp120-binding Ab in the passively transferred immune IgG decayed with a half-life of ∼2 wk (data not shown). The control macaques failed to develop any gp120-specific binding Abs following infection with the exception of macaque 36455 that exhibited a binding titer of 240–270 at wk 8–10 postchallenge.

Examination of ADCC activity in the macaque plasma postchallenge revealed an initial drop in ADCC titer as measured using SIV gp120-coated target cells (Fig. 2⇓, A and B). This paralleled the decay of the transferred immune IgG. However, at wk 4, the ADCC titers were boosted and reached peak titers at 6 wk postchallenge. Similarly, ADCC activity against gp120-coated target cells appeared at wk 4 in the macaques that received control IgG and peaked at wk 6. These results are consistent with induction of de novo ADCC activity resulting from SIV infection.

FIGURE 2.
FIGURE 2.

Titers of ADCC-mediating Abs in sequential plasma specimens from neonatal macaques using SIVgp120-coated CEM.NKr cells (A and B) and H9 cells infected with SIVmac251 (C and D) as targets. Recipients of immune IgG (A and C); control IgG (B and D). Values for wk 9, 10, and 11 are combined as in Fig. 1⇑.

ADCC activity against SIVmac251-infected cells exhibited a similar pattern (Fig. 2⇑, C and D). In macaques that received immune IgG, the ADCC titers declined over the first 2 wk and then were boosted at wk 4. In the control animals, de novo ADCC activity again appeared at wk 4. When assessed by either gp120-coated targets or SIV-infected targets, ADCC titers remained 10- to 100-fold higher in the macaques that received immune IgG compared with the controls, reflecting maintenance of ADCC activity in vivo from the passively transferred IgG in addition to de novo activity resulting from SIV infection. Using SIV-infected targets, peak titers were observed at wk 4 postchallenge, with the experimental group exhibiting a geometric mean ADCC titer of 98,704, and the controls a geometric mean titer of 7,396. Using gp120-coated targets, peak titers were observed at wk 6 postchallenge, with geometric means of 173,564 and 1,339 for the experimental and control groups, respectively. As the immune IgG continued to decay and as disease progression occurred, the ADCC activity observed in plasma samples gradually declined until the time of euthanasia.

We also monitored ADCVI activity in the macaque plasmas at 4 wk postchallenge. The results are similar to those for ADCC activity in showing maintenance of ADCVI activity at wk 4 in the immune IgG group and induction of de novo ADCVI activity in the control group (Table III⇓). Peak ADCVI activity remained significantly higher at wk 4 postchallenge in the macaques that received immune IgG (p = 0.0087). Overall, however, ADCVI titers were low, and despite the maintenance of activity, titers were not boosted in the experimental animals.

View this table:
Table III.

Evaluation of ADCVI in plasma from experimental and control macaques, pre- and post-oral SIVmac251 challenge

In conjunction with induction of de novo ADCC and ADCVI activity resulting from SIV infection, the macaques also developed neutralizing Abs (Fig. 3⇓). By wk 4 and 6 postchallenge, two of six macaques that received control IgG had developed low titer Abs able to achieve 90% neutralization of the SIVmac251 challenge virus, whereas in the group that received immune IgG, three of six developed such Abs. If a 75% end point was used for determining titer, five macaques in each group were able to neutralize the primary SIVmac251 stock. Overall, the neutralizing Ab titers observed following infection were similar between the experimental and control macaques, which is consistent with the observed lack of neutralizing Ab prechallenge in the immune IgG.

FIGURE 3.
FIGURE 3.

Development of neutralizing Abs against the SIVmac251 challenge virus following oral administration to the neonatal macaques. Reciprocal titers are shown for two end points. ∗, An equivocal result was obtained for this macaque: one titer < 25 and one titer of 55. Sufficient plasma was not available for a third repetition. The limit of detection was 1:25; reciprocal titers < 25 are plotted as 25. 

Overall, the spectrum of Abs induced or boosted at 4 wk postchallenge as a result of SIV infection did not exert any effect on the challenge outcomes. Viral loads were not diminished, and CD4 counts did not show any consistent increase at wk 4–6.

NK cells in the infant macaques

In view of the ADCC and ADCVI activities mediated by the macaque plasma samples in vitro, but the lack of any observable effect on challenge outcome in vivo, we considered the levels of potential ADCC effector cells in the blood of the infant macaques before challenge. γδT cells were not measured; however, both neutrophils and monocytes were present at adequate levels (8159 ± 565 and 960 ± 132/μl, respectively). In contrast, the absolute mean numbers and percentages (Fig. 4⇓) of NK cells in both macaque groups were low at the time of challenge and subsequently increased by 2–4 wk. The percentages observed were similar to those reported for healthy rhesus neonates up to 3 wk of age (30) and for SIV-infected infant macaques (31) but lower than the 9–13% reported for juvenile macaques. In older rhesus macaques, NK cells peak ∼2 wk post-SIV infection before returning to prechallenge levels (33), and a similar pattern was observed here. Unfortunately, blood volumes were too small to investigate NK functionality.

FIGURE 4.
FIGURE 4.

Absolute numbers (A) and percentages of NK cells (B) in the macaques following oral challenge with SIVmac251. Week 0 is equivalent to 2–6 days of age. Values shown are means ± SEM. 

Discussion

As current vaccine candidates have not induced broadly neutralizing Abs able to provide “sterilizing immunity” against HIV infection, protection that diminishes the initial viral burden to a level low enough to provide prolonged viremia control, slow disease progression, and avoidance of virus transmission has become the goal of vaccine design. In this regard, if potent, vaccine-induced Abs are present before HIV exposure, the ADCC mechanism could provide a rapid response since initial expansion of a specific T cell population is not required. The ADCC mechanism targets virus-infected cells, but ADCC-mediating Abs might also prevent infection by rapidly interacting with virus bound to CD4 target cells, leading to cell lysis by ADCC before virus entry. Systemic Abs in underlying mucosal tissues might contribute to protection from mucosal challenge by blocking expansion of the founder seed stocks of virus-infected cells that lead to systemic infection (34). Neutralizing mAbs and immune IgG shown to mediate postexposure prophylaxis (12, 13) might also protect in part by an ADCC mechanism. Delay of Ab treatment as much as 6 h after initial virus exposure has blocked subsequent infection (13). While neutralizing activity could block cell-to-cell virus transmission, an ADCC mechanism could eliminate cells expressing target Ags before release of infectious virions. The ability of potent neutralizing mAbs to mediate ADCC activity has not been explored systematically, although 2G12 and IgG1 b12 both possess such ability (23, 35, 36).

In the present study, we addressed the protective efficacy of ADCC activity apart from neutralization, using nonneutralizing Ab previously shown to mediate ADCC activity that correlated with reduced acute viremia following SIVmac251 intrarectal challenge (6, 9). Initial in vitro characterization of the pooled immune serum revealed a complexity in the epitope(s) recognized by the ADCC-mediating Abs. ADCC Ab titers against gp120-coated targets were higher than Ab binding titers against gp120 (Table I⇑). This suggests that when gp120 was bound to CD4 on the target cell, a conformational change exposed alternate epitopes recognized by the immune sera. This speculation is supported by the observation that animals that received control IgG developed de novo ADCC activity against gp120-coated target cells following SIV infection (Fig. 2⇑D) but continued to be negative for gp120 binding Ab (data not shown). Apparently, similar conformational epitopes appear upon SIV infection and induce Abs able to mediate ADCC killing of gp120-coated targets. It is also possible that ADCC is simply more sensitive than ELISA, as suggested by Sawyer et al. (1). However, in contrast to our results, this greater sensitivity was obtained using different Ags for evaluating binding (Dupont HIV ELISA) and ADCC activity (vaccinia-HIV gp120-infected targets).

Our results show clearly that the immune IgG infused did not protect newborn rhesus macaques from oral SIV infection as shown by viral burdens, disease progression, and survival times similar to those of neonates that received control IgG. Several factors may explain the difference between the previously observed correlation and the challenge outcome reported here.

First, a high challenge dose was administered to ensure infection of the control neonates. The previous correlation of ADCC activity with reduced acute viremia was observed following an intrarectal challenge with ∼500 TCID50 of SIVmac251 (9), while in the present study, the neonates received two sequential oral doses of 105 TCID50 SIVmac251 each. Recent studies in neonatal rhesus macaques (18) have used repeated lower-dose oral exposures of 104 TCID50 each (2 ml of 7 × 107 viral RNA copies/ml), which is still high compared with average levels of SIV in breast milk during chronic SIV infection (2 × 102–2 × 103 viral RNA copies/ml; Ref. 37) but more relevant to natural infection. However, in the present study, insufficient immune IgG was available for continuous transfer over several days of repeated low-dose challenges. Thus, the 400-fold greater dose used in comparison to the previous study may have obscured any protection mediated by the immune IgG.

Second, the route of challenge may have influenced the outcome. Following oral infection of juvenile and neonatal macaques, SIV quickly accesses draining lymph nodes and rapidly spreads to tissues throughout the body within 1–2 days (38). In contrast, while some rapid dissemination of SIV to distal sites occurs following vaginal exposure, systemic infection is delayed ∼6 days by establishment of small “founder” populations of infected cells in the cervicovaginal mucosa, which then spread SIV throughout the lymphoid tissues (39). Thus, there may be a greater window of time for immune intervention to control viral replication and spread. Whether rectal SIV transmission and dissemination is similar to vaginal infection has not been well investigated, nor have various challenge routes been directly compared. Nevertheless, it is plausible that the kinetics of oral infection may be more rapid due to more rapid transfer of virus to the lymphatic system. If so, protection would be easier to achieve following rectal transmission, explaining the positive correlation with ADCC activity in the earlier study and the lack of protection mediated by anti-envelope Abs observed here.

The distribution of the passively transferred immune IgG in tissue/mucosal compartments after s.c. administration is also not known. ADCC-mediating Ab was present in plasma of the neonates at the time of oral challenge. Whether Ab was also present in the oral mucosa and at a level comparable to that in the rectal mucosa in the earlier study is not known. Rectal secretions in the earlier study were negative for ADCC activity (6). However, consistent with the presence of low-titered SIV-specific Ab in saliva following passive transfer of SIV hyperimmune serum (15), low-titered ADCC activity against gp120-coated and SIVmac251-infected target cells (titers of ∼25 and ranging from <5 to 20, respectively) was present in saliva of the experimental macaques 1 wk postchallenge. These titers declined over the next 3 wk, whereas saliva samples of the control neonates were consistently negative.

Third, the neonatal macaques may have lacked sufficient and fully functional NK effector cells at the time of challenge. NK cells are major effector cells that mediate ADCC and related ADCVI activities. Monocyte/macrophages also mediate these activities and, in older animals, are probably the main effector cells for ADCVI (8). The contribution of other cell types to ADCC and ADCVI effector function in neonatal macaques is not known. In macaques, as in humans, the proportion of NK cells increases with age. Neonatal macaques have ∼3.5% NK cells in peripheral blood (31) in contrast to ∼13% in adults (40). The mean percentage of NK cells in the neonates at challenge was as expected, low (3.5%), and declined following SIV infection instead of exhibiting the expected age-dependent increase (Fig. 4⇑). It is possible that NK cells were insufficient for potent ADCC activity in vivo. A significant negative correlation among all 12 neonates between NK cell percentage and viral load was observed at wk 1 postchallenge (p < 0.01), the first time point at which viremia was measured. This correlation disappeared at later time points but suggests that NK cell number may impact the outcome of viral exposures.

Additionally, human infants are less able to mediate ADCC against HIV-infected cells than adults (41). Blood volumes obtained from the rhesus neonates were not sufficient for assessment of NK functionality. However, control of SIV infection by the ADCC mechanism postchallenge may have been impaired in the neonatal macaques.

Fourth, too little immune IgG may have been passively transferred. Based on anti-envelope binding titers, plasma Ab concentrations at challenge were ∼13-fold lower than that in the original sera/plasma pool. No benchmarks for protective ADCC levels have been established. The mean net ADCC killing of SIV-infected targets in the previous study in which ADCC activity was correlated with reduced acute viremia was 31.1% (6), whereas in the present study, net ADCC killing mediated by plasma from the experimental neonates was 12.4%. The 2.5-fold lesser activity may have been below the threshold at which protection could be detected. ADCVI activity was also low at challenge in comparison to plasma from infected macaques.

Finally, ADCC activity alone may not be adequately protective. In the previous study, although the immunized macaques did not possess neutralizing Abs, they exhibited strong cellular immune responses (9, 42, 43). It is possible that ADCC activity, in conjunction with cellular immunity, acted to partially control acute infection. Such adaptive cellular immunity was of course not present in the neonatal macaques. While confirmation of the role of individual immune parameters in protection is informative, in vivo a single protective correlate may not exist. Rather, the sum total of immune responses may ultimately control infectious diseases.

Overall, the passively transferred immune IgG did not protect the neonatal macaques from oral SIVmac251 challenge in this pilot experiment. However, the results suggest a design for further studies exploring the potential role in protection of nonneutralizing but ADCC-mediating Abs. Passive transfer experiments should be conducted in juvenile or adult animal models, using more potent IgG preparations and exploring additional routes of virus transmission that allow lower challenge doses. ADCC remains a potentially important mechanism for vaccine development, spanning innate and adaptive immunity. The extent to which it can contribute to protective efficacy should be clearly defined.

Acknowledgments

We thank William Sutton and members of the Immunology Core, Humoral Immunity Subcore of the University of Washington Center for AIDS Research, Seattle Biomedical Research Institute for IgG purification; Stephen Whitney for ELISA binding assays; Emily Blackwood, Kimberly Schmidt, and the veterinary staff, Colony Services and Clinical Laboratory of the California National Primate Research Center for their technical assistance. The following reagent was obtained from the AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases, National Institutes of Health: CEM-NKr cells from Dr. Peter Cresswell.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported in part by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, by National Institutes of Health Grant R01 AI52039 (to D.N.F.), and by a National Center for Research Resources supplement to the California National Primate Research Center Base Operating Grant (RR00169).

  • ↵2 Address correspondence and reprint requests to Dr. Marjorie Robert-Guroff, National Institutes of Health, National Cancer Institute, 41 Medlars Drive, Building 41, Room D804, Bethesda, MD 20892-5065. E-mail address: guroffm{at}mail.nih.gov

  • ↵3 Abbreviations used in this paper: ADCC, Ab-dependent cellular cytotoxicity; ADCVI, Ab-dependent cell-mediated virus inhibition; Ad, adenovirus; Ad5hr, Ad type 5 host range mutant; TCID50, 50% inhibiting tissue culture-infective dose; RFADCC, rapid fluorometric ADCC.

  • Received April 5, 2006.
  • Accepted July 3, 2006.

References

  1. ↵
    Sawyer, L. A., D. A. Katzenstein, R. M. Hendry, E. J. Boone, L. K. Vujcic, C. C. Williams, S. L. Zeger, A. J. Saah, C. R. Rinaldo, Jr, J. P. Phair, et al 1990. Possible beneficial effects of neutralizing antibodies and antibody-dependent cell mediated cytotoxicity in human immunodeficiency virus infection. AIDS Res. Hum. Retroviruses 6: 341-356.
    OpenUrlCrossRefPubMed
  2. ↵
    Tyler, D. S., H. K. Lyerly, K. J. Weinhold. 1989. Anti-HIV 1 ADCC. AIDS Res. Hum. Retroviruses 5: 557-563.
    OpenUrlCrossRefPubMed
  3. ↵
    Ahmad, A., J. Menezes. 1996. Antibody-dependent cellular cytotoxicity in HIV infections. FASEB J. 10: 258-266.
    OpenUrlAbstract
  4. ↵
    Forthal, D. N., G. Landucci, B. Keenan. 2001. Relationship between antibody dependent cellular cytotoxicity, plasma HIV type 1 RNA and CD4+ lymphocyte count. AIDS Res. Hum. Retroviruses 6: 553-561.
    OpenUrl
  5. ↵
    Banks, N. D., N. Kinsey, J. Clements, J. E. K. Hildreth. 2002. Sustained antibody-dependent cell-mediated cytoxicity (ADCC) in SIV-infected macaques correlates with delayed progression to AIDS. AIDS Res. Hum. Retroviruses 18: 1197-1205.
    OpenUrlCrossRefPubMed
  6. ↵
    Gomez-Roman, V. R., L. J. Patterson, D. Venzon, D. Leiwehr, K. Aldrich, R. Florese, M. Robert-Guroff. 2005. Vaccine elicited antibodies mediate antibody-dependent cellular cytotoxicity correlated with significantly reduced acute viremia in rhesus macaques challenged with SIVmac251. J. Immunol. 174: 2185-2189.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Forthal, D., G. Landucci, M. Marthas, K. Van Rompay. 2005. Non-neutralizing antibody that prevents SIV infection in neonatal rhesus macaques (RHMS) potently inhibits SIV in an antibody-dependent cell-mediated virus inhibition (ADCVI) assay. AIDS Vaccine Meeting, September 6–9 50 , Montreal, Quebec, Canada.
  8. ↵
    Forthal, D. N., G. Landucci, E. S. Daar. 2001. Antibody from patients with acute human immunodeficiency virus (HIV) infection inhibits primary strains of HIV type 1 in the presence of natural-killer effector cells. J. Virol. 75: 6953-6961.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Patterson, L. J., N. Malkevitch, J. Pinczewski, V. R. Gomez-Roman, L. Wang, V. S. Kalyanaraman, P. D. Markham, F. A. Robey, M. Robert-Guroff. 2004. Protection against mucosal simian immunodeficiency virus SIVmac251 challenge by using replicating adenovirus-SIV multigene vaccine priming and subunit boosting. J. Virol. 78: 2212-2221.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Mascola, J. R., M. G. Lewis, G. Stiegler, D. Harris, T. C. VanCott, D. Hayes, M. K. Louder, C. R. Brown, C. V. Sapan, S. S. Frankel, et al 1999. Protection of macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies. J. Virol. 73: 4009-4018.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Parren, P. W., P. A. Marx, A. J. Hessell, A. Luckay, J. Harouse, C. Cheng-Mayer, J. P. Moore, D. R. Burton. 2001. Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro. J. Virol. 75: 8340-8347.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Ferrantelli, F., R. A. Rasmussen, K. A. Buckley, P-L. Li, T. Wang, D.C. Montefiori, H. Katinger, G. Stiegler, D. C. Anderson, H. M. McClure, R. M. Ruprecht. 2004. Complete protection of neonatal rhesus macaques against oral exposure to pathogenic simian-human immunodeficiency virus by human anti-HIV monoclonal antibodies. J. Infect. Dis. 189: 2167-2173.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Nishimura, Y., T. Igarashi, N. L. Haigwood, R. Sadjadpour, O. K. Donau, C. Buckler, R. J. Plishka, A. Buckler-White, M. A. Martin. 2003. Transfer of neutralizing IgG to macaques 6 h by not 24 h after SHIV infection confers sterilizing protection: implications for HIV-1 vaccine development. Proc. Natl. Acad. Sci. USA 100: 15131-15136.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Haigwood, N. L., D. Montefiori, W. F. Sutton, J. McClure, A. J. Watson, G. Voss, V. M. Hirsch, B. A. Richardson, N. L. Letvin, S. L. Hu, P. R. Johnson. 2004. Passive immunotherapy in simian immunodeficiency virus-infected macaques accelerates the development of neutralizing antibodies. J. Virol. 78: 5983-5995.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Van Rompay, K. K. A., C. J. Berardi, S. Dillard-Telm, R. P. Tarara, D. R. Canfield, C. R. Valverde, D. C. Montefiori, K. Stefano Cole, R. C. Montelaro, C. J. Miller, M. L. Marthas. 1998. Passive immunization of newborn rhesus macaques prevents oral simian immunodeficiency virus infection. J. Infect. Dis. 177: 1247-1259.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Van Rompay, K. K.. 2005. Antiretroviral drug studies in nonhuman primates: a valid animal model for innovative drug efficacy and pathogenesis experiments. AIDS Rev. 7: 67-83.
    OpenUrlPubMed
  17. ↵
    Van Rompay, K. K. A., P. J. Dailey, R. P. Tarara, D. R. Canfield, N. L. Aguirre, J. M. Cherrington, P. D. Lamy, N. Bischofberger, N. C. Pedersen, M. L. Marthas. 1999. Early short-term 9-[2-R-(Phosphonomethoxy)propyl]adenine treatment favorably alters the subsequent disease course in simian immunodeficiency virus-infected newborn rhesus macaques. J. Virol. 73: 2947-2955.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Van Rompay, K. K. A., K. Abel, J. R. Lawson, R. P. Singh, K. A. Schmidt, T. Evans, P. Earl, D. Harvey, G. Franchini, J. Tartaglia, et al 2005. Attenuated poxvirus-based simian immunodeficiency virus (SIV) vaccines given in infancy partially protect infant and juvenile macaques against repeated oral challenge with virulent SIV. J. Acquir. Immune Defic. Syndr. 38: 124-134.
    OpenUrlCrossRefPubMed
  19. ↵
    Zhao, J., J. Pinczewski, V. R. Gomez-Roman, D. Venzon, V. S. Kalyanaraman, P. D. Markham, K. Aldrich, M. Moake, D. C. Montefiori, Y. Lou, et al 2003. Improved protection of rhesus macaques against intrarectal simian immunodeficiency virus SIVmac251 challenge by a replication competent Ad5hr-SIVenv/rev and Ad5hr-SIVgag recombinant priming/gp120 boosting regimen. J. Virol. 77: 8354-8365.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    National Research Council. 1996. Guide for the Care and Use of Laboratory Animals National Academy Press, Washington, DC.
  21. ↵
    Van Rompay, K. K., J. L. Greenier, K. S. Cole, P. Earl, B. Moss, J. D. Steckbeck, B. Pahar, T. Rourke, R. C. Montelaro, D. R. Canfield, et al 2003. Immunization of newborn rhesus macaques with simian immunodeficiency virus vaccines prolongs survival after oral challenge with virulent SIVmac251. J. Virol. 77: 179-190.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Van Rompay, K. K., R. P. Singh, B. Pahar, D. L. Sodora, C. Wingfield, J. R. Lawson, M. R. Marthas, N. Bischofberger. 2004. CD8+ cell mediated suppression of virulent simian immunodeficiency virus during tenofovir treatment. J. Virol. 78: 5324-5337.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Gomez-Roman, V. R., R. H. Florese, L. J. Patterson, B. Peng, D. Venzon, K. Aldrich, M. Robert-Guroff. 2006. A simplified method for the rapid fluorometric assessment of antibody-dependent cell mediated cytotoxicity. J. Immunol. Methods 308: 53-67.
    OpenUrlCrossRefPubMed
  24. ↵
    Forthal, D. N., G. Landucci. 1998. In vitro reduction of virus infectivity by antibody-dependent cell-mediated immunity. J. Immunol. Methods 220: 129-138.
    OpenUrlCrossRefPubMed
  25. ↵
    Buge, S. L., E. Richardson, S. Alipanah, P. Markham, S. Chen, N. Kalyan, C. J. Miller, M. Lubeck, S. Udem, J. Eldridge, M. Robert-Guroff. 1997. An adenovirus-SIV env vaccine elicits humoral, cellular and mucosal immune responses in rhesus macaques and decreases viral burden following vaginal challenge. J. Virol. 71: 8531-8541.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    Otsyula, M., C. Miller, M. L. Marthas, K. K. Van Rompay, J. Collins, N. Pedersen, M. McChesney. 1996. Virus-induced immunosuppression is linked to rapidly fatal disease in infant rhesus macaques infected with simian immunodeficiency virus. Pediatr. Res. 39: 630-635.
    OpenUrlCrossRefPubMed
  27. ↵
    Chackerian, B., N. L. Haigwood, J. Overbaugh. 1995. Characterization of a CD4-expressing macaque cell line that can detect virus after a single replication cycle and can be infected by diverse simian immunodeficiency virus isolates. Virology 213: 386-394.
    OpenUrlCrossRefPubMed
  28. ↵
    Mahalanabis, M., V. M. Hirsch, N. L. Haigwood. 2005. Infection with a molecularly cloned SIVsm virus elicits high titer homologous neutralizing antibodies with heterologous neutralizing activity. J. Med. Primatol. 34: 253-261.
    OpenUrlCrossRefPubMed
  29. ↵
    Weinhold, K J.. 1990. Nonrestricted forms of anti-HIV-1 cytotoxicity. A. Aldovini, Jr, and B. D. Walker, Jr, eds. Techniques in HIV Research 187-199. Stockton Press, New York.
  30. ↵
    DeMaria, M. A., M. Casto, M. O’Connell, R. P. Johnson, M. Rosenzweig. 2000. Characterization of lymphocyte subsets in rhesus macaques during the first year of life. Eur. J. Haematol. 65: 245-257.
    OpenUrlCrossRefPubMed
  31. ↵
    Van Rompay, K. K. A., R. P. Singh, L. L. Brignolo, J. R. Lawson, K. A. Schnidt, B. Pahar, D. R. Canfield, R. P. Tarara, D. L. Sodora, N. Bischofberger, M. L. Marthas. 2004. The clinical benefits of tenofovir for simian immunodeficiency virus-infected macaques are larger than predicted by its effects on standard viral and immunologic parameters. J. Acquir. Immune Defic. Syndr. 36: 900-914.
    OpenUrlCrossRefPubMed
  32. ↵
    Pal, R., D. Venzon, N. L. Letvin, S. Santra, D. C. Montefiori, N. R. Miller, E. Tryniszewska, M. G. Lewis, T. C. VanCott, V. Hirsch, et al 2002. ALVAC-SIV gag-pol-env-based vaccination and macaque major histocompatibility complex class I (A*01) delay simian immunodeficiency virus SIVmac-induced immunodeficiency. J. Virol. 76: 292-302.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Benlhassan-Chahour, K., C. Penit, V. Dioszehgy, F. Vasseur, G. Janvier, Y. Riviere, N. Dereuddre-Bosquet, D. Dormont, R. Le Grand, B. Vaslin. 2003. Kinetics of lymphocyte proliferation during primary immune response in macaques infected with pathogenic simian immunodeficiency virus SIVmac251: preliminary report of the effect of early antiviral therapy. J. Virol. 77: 12479-12493.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Haase, A. T.. 2005. Perils at mucosal front lines for HIV and SIV and their hosts. Nat. Rev. Immunol. 5: 783-792.
    OpenUrlCrossRefPubMed
  35. ↵
    Trkola, A., M. Purtscher, T. Muster, C. Ballaun, A. Buchacher, N. Sullivan, K. Srinivasan, J. Sodroski, J. P. Moore, H. Katinger. 1996. Human mono-clonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1. J. Virol. 70: 1100-1108.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Hezareh, M., A. J. Hessell, R. C. Jensen, J. G. J. Van de Winkel, P. W. H. I. Parren. 2001. Effector function activities of a panel of mutants of a broadly neutralizing antibody against human immunodeficiency virus type 1. J. Virol. 75: 12161-12168.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Amedee, A. M., J. Rychert, N. Lacour, L. Fresh, M. Ratterree. 2004. Viral and immunological factors associated with breast milk transmission of SIV in rhesus macaques. Retrovirology 1: 17
    OpenUrlCrossRefPubMed
  38. ↵
    Milush, J. M., D. Kosub, M. Marthas, K. Schmidt, F. Scott, A. Wozniakowski, C. Brown, S. Westmoreland, D. L. Sodora. 2004. Rapid dissemination of SIV following oral inoculation. AIDS 18: 2371-2380.
    OpenUrlPubMed
  39. ↵
    Miller, C. J., Q. Li, K. Abel, E.-Y. Kim, Z.-M. Ma, S. Wietgrefe, L. La Franco-Scheuch, L. Compton, L. Duan, M. Dykhuizen Shore, et al 2005. Propagation and dissemination of infection after vaginal transmission of simian immunodeficiency virus. J. Virol. 79: 9217-9227.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    Rappocciolo, G., J. S. Allan, J. W. Eichberg, T. C. Chanh. 1992. A comparative study of natural killer cell activity, lymphoproliferation, and cell phenotypes in nonhuman primates. Vet. Pathol. 29: 53-59.
    OpenUrlCrossRefPubMed
  41. ↵
    Merrill, J. D., M. Sigaroudinia, S. Kohl. 1996. Characterization of natural killer and antibody-dependent cellular cytotoxicity of preterm infants against human immunodeficiency virus-infected cells. Pediatr. Res. 40: 498-503.
    OpenUrlCrossRefPubMed
  42. ↵
    Patterson, L. J., N. Malkevitch, J. Pinczewski, D. Venzon, Y. Lou, B. Peng, C. Munch, M. Leonard, E. Richardson, K. Aldrich, et al 2003. Potent, persistent induction and modulation of cellular immune responses in rhesus macaques primed with Ad5hr-simian immunodeficiency virus (SIV) env/rev, gag, and/or nef vaccines and boosted with SIV gp120. J. Virol. 77: 8607-8620.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    Malkevitch, N., L. J. Patterson, K. Aldrich, E. Richardson, W. G. Alvord, M. Robert-Guroff. 2003. A replication competent Ad5hr-SIV recombinant priming/subunit protein boosting vaccine regimen induces broad, persistent SIV-specific cellular immunity to dominant and subdominant epitopes in mamu-A*01 rhesus macaques. J. Immunol. 170: 4281-4289.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section