Hyperimmune Bovine Colostrum as a Low-Cost, Large-Scale Source of Antibodies with Broad Neutralizing Activity for HIV-1 Envelope with Potential Use in Microbicides

Abstract

Bovine colostrum (first milk) contains very high concentrations of IgG, and on average 1 kg (500 g/liter) of IgG can be harvested from each immunized cow immediately after calving. We used a modified vaccination strategy together with established production systems from the dairy food industry for the large-scale manufacture of broadly neutralizing HIV-1 IgG. This approach provides a low-cost mucosal HIV preventive agent potentially suitable for a topical microbicide. Four cows were vaccinated pre- and/or postconception with recombinant HIV-1 gp140 envelope (Env) oligomers of clade B or A, B, and C. Colostrum and purified colostrum IgG were assessed for cross-clade binding and neutralization against a panel of 27 Env-pseudotyped reporter viruses. Vaccination elicited high anti-gp140 IgG titers in serum and colostrum with reciprocal endpoint titers of up to 1 × 10⁵. While nonimmune colostrum showed some intrinsic neutralizing activity, colostrum from 2 cows receiving a longer-duration vaccination regimen demonstrated broad HIV-1-neutralizing activity. Colostrum-purified polyclonal IgG retained gp140 reactivity and neutralization activity and blocked the binding of the b12 monoclonal antibody to gp140, showing specificity for the CD4 binding site. Colostrum-derived anti-HIV antibodies offer a cost-effective option for preparing the substantial quantities of broadly neutralizing antibodies that would be needed in a low-cost topical combination HIV-1 microbicide.

INTRODUCTION

In the absence of an effective prophylactic vaccine against human immunodeficiency virus type 1 (HIV-1), there is an urgent need for female-controlled, safe, effective, and inexpensive biomedical preventions, such as topical microbicides for the prevention of sexually transmitted HIV-1 infections (16, 34, 37). Despite the failure of previous microbicide trials (14, 51, 58, 59), the antiretroviral drug tenofovir demonstrated significant reduction in HIV acquisitions by 39% if used topically in a 1% gel (CAPRISA 004 trial) (24) but did not provide any protection if used orally (VOICE trial) (36). Irrespective of differences in outcome resulting from delivery modality and adherence, the use of antiretroviral drugs as microbicides is controversial in developing countries, where affordability and accessibility to antiretroviral drugs are extremely limited. Further, the use of current therapeutic drugs for prophylaxis may increase the selection pressure for drug-resistant HIV escape mutants. Maximum microbicide potency is likely to require combination microbicides incorporating different components, such as antibodies (Abs) capable of blocking HIV infection.

Broad and potent neutralizing Abs (NAbs), primarily raised against the envelope protein (Env), have been isolated from the serum of HIV-1-infected individuals. These monoclonal NAbs (mNAbs) bind to conserved functional epitopes on the gp140 Env: b12 and VRC01 targeting the CD4 binding site, 2G12 targeting glycan, 2F5 and 4E10 targeting the membrane proximal region, 447-52D targeting a CD4-induced face, and PG9/16, targeting oligomeric V3 structures (5, 7, 13, 61, 63). mNAbs targeting the membrane proximal regions are broad acting, with 2F5 and 4E10 neutralizing 67 and 100%, respectively, of a panel of 90 divergent viruses (5), indicating strongly conserved epitopes. The CD4 binding site targeting mNAb b12 neutralized 50% of the viruses in the same panel, and 2G12 neutralized 41%. 447-52D was less broad acting, neutralizing 19% of viruses. The VRC01 mNAb, which also targets the CD4 binding site, neutralized 91% of a 190-virus panel (63), indicating a high degree of conservation of the contact residues of the Env epitope for this antibody.

Intravenous and vaginal application of patient-derived anti-HIV-1 immunoglobulin and/or mNAbs 2G12, 2F5, and b12 can afford dose-dependent sterile protection to primates from intravenous or vaginal challenge with chimeric simian-human immunodeficiency virus (SHIV) (32, 33, 45). Protection correlated with NAb concentration and in vitro neutralizing activity (21, 32, 33, 40, 45, 47, 60). In contrast, a nonneutralizing variant of b12 did not provide protection in primates (8).

High concentrations of mNAbs show promise for microbicide formulations, but they are currently prohibitively expensive to produce in the large amounts required. An alternative source for low-cost HIV-1-specific NAbs is bovine colostrum (BC). BC is highly enriched with maternal immunoglobulin that is actively drawn from the serum. The most abundant immunoglobulins in BC are IgG, with up to 50 mg/ml (primarily IgG1), but IgA and IgM are also present (up to 4 mg/ml) (55). BC also contains antimicrobial peptides and proteins, such as lactoferrin lactoperoxidase and lysozyme, that can stimulate innate antiviral pathways and adaptive immune responses (52, 55, 57). Vaccination of cows against specific pathogens results in polyclonal pathogen-specific Abs in BC (hyperimmune BC [HBC]). Purified HBC Abs have successfully been used for the oral treatment of dental caries (26) and a variety of gastrointestinal infections caused by pathogenic bacteria (20, 25, 46, 56) or virus (27, 35, 39).

Here, we developed a vaccination strategy in cows using oligomeric HIV-1 gp140 antigens that induced high titers of anti-gp140 Abs in HBC. Both HBC and purified HBC IgG showed broad neutralizing activity against a diverse array of Env-pseudotyped reporter viruses. This approach provides low-cost production of HIV-1 NAbs for a potentially efficacious and affordable microbicide.

MATERIALS AND METHODS

Production and purification of oligomeric gp140.

Oligomeric gp140 of Env truncated at the membrane proximal external region was created by stably transfecting pN1-UG8-140 and pN1-MW-140 (the 92UG037.8 and 93MW965.26 strains, respectively; provided by F. Gao and B.H. Hahn) into 293T cells and pN1-AD8-140 (AD8 clone of ADA; provided by M. Martin) into HeLa cells as previously described (11). Secreted gp140 was purified from cell culture supernatants by lentil-lectin affinity chromatography and size exclusion chromatography using a 16/60 Superdex 200 column as previously described (10). Fractions enriched for gp140 trimers were pooled and concentrated for use in vaccinations.

Vaccination of cows.

Vaccination of four female cows was performed with Ethics Committee approval [POCTAA (3)003 A04]. Each vaccination consisted of 100 μg of purified gp140 oligomers in adjuvant (Montanide ISO 206; Seppic, France) injected intramuscularly into the flank (vaccination regime) (Fig. 1A). Primary vaccination took place either before conception (group NP) or during the second trimester of pregnancy (group P) followed by three or two boost vaccinations after conception, respectively. Final vaccination was given approximately 4 weeks before giving birth. Within each group, one animal was vaccinated either with clade B (AD8) only (B) or with equal amounts of clade A (UG8), B (AD8), and C (MW) gp140 (33.33 μg each) (trimix). Blood samples were collected into serum tubes (Venoject glass tubes; Venoject) at regular intervals, avoiding the period 4 weeks before birth to reduce the risk of miscarriage. Blood was centrifuged at 1,300 × g for 12 min at room temperature. Serum was removed and stored in aliquots at −80°C. HBC was collected within 6 h postpartum by milking and was stored at −20°C prior to use.

Fig 1.

Vaccination regime and gp140-specific IgG titers. (A) Primary vaccination was initiated during the second trimester of pregnancy (P cows, n = 2) or prior to conception (NP cows, n = 2). Cows received 100 μg of gp140 Env oligomers consisting of either clade B alone or a mixture of clade A, B, and C (trimix). (B) Reciprocal Env-specific IgG endpoint titers in serum against gp140 AD8 over the time of vaccination. Arrows indicate additional boost vaccinations; results show the means from 3 repeats. (C) Serum IgG titers 9 weeks after primary vaccination. (D) Colostrum IgG titers specific against clade A (UG8), B (AD8), and C (MW) gp140 Env; results show the means from 3 repeats; error bars represent the standard deviation (SD). (E) gp140-specific binding of purified colostrum IgG against clade A, B, and C gp140 Env. Endpoint concentrations are listed in the table below the graphs. Reciprocal IgG endpoint titers were determined by ELISA against the respective gp140 Env proteins, and a positive signal was defined as an OD of >2-fold of preimmunization samples, nonimmune colostrum, or nonimmune purified IgG.

Preparation of colostrum and purified colostrum IgG.

BC was defatted and pasteurized by centrifugation at 10,000 × g for 30 min at 4°C, incubation at 63°C for 30 min, and further centrifugation at 10,000 × g for 10 min. For IgG purification, casein-depleted colostral whey was prepared by adjusting the pH to 4.6 with an equal volume of 0.2 M sodium acetate solution (pH 4.0) while mixing at 37°C for 2 h followed by cooling and centrifugation at 10,000 × g for 30 min. Colostral whey was adjusted to pH 6.6 and then dialyzed against phosphate-buffered saline (PBS) using a 30-kDa cutoff ultrafiltration membrane (Amicon Ultra, 15 ml; Millipore). IgG was purified by protein G Sepharose chromatography (GE Healthcare). After elution with 50 mM citrate, pH 2.6, IgG-containing fractions were neutralized to pH 7.0 by the addition of 1 M Tris, pH 8.0, and then dialyzed against PBS. Purified IgG was filter sterilized, and concentrations were measured by reading absorbance at 280 nm.

Anti-bovine IgG ELISA.

gp140-specific IgG binding titers in sera and whole HBC and purified HBC IgGs were measured by enzyme-linked immunosorbent assay (ELISA). Purified gp140 in 100 μl/well coating buffer (pH 9.8; 2 mM Tris, 10 mM NaCl) was coated onto 96-well polyvinyl flat-bottom plates (Pathtech, Australia) at 100 ng/well overnight at 4°C. All subsequent steps were performed at room temperature. Wells were washed sequentially in PBST (PBS-0.1% Tween 20) and PBS and then blocked with 5% casein in PBS for 2 h. Wells were washed as described above, followed by the addition of half-log dilutions of sera or HBC or 2-fold dilutions of purified HBC IgG in 5% casein. After 4 h of incubation, wells were washed and horseradish peroxidase (HRP)-conjugated rabbit anti-bovine IgG antibody (Sigma) was added and incubated for 1 h. Color reactions were developed using 3,3′-5,5′-tetramethylbenzidine (TMB), and absorbance was measured at 405 nm against a reference of 492 nm. A positive IgG signal was defined as an optical density (OD) of >2-fold higher than that obtained with either serum samples taken prior to vaccination, whole nonimmune BC, or IgG purified from nonimmune BC, as appropriate. IgG titers are presented as averages from 3 repeats.

Competition ELISA.

To determine purified HBC IgG-specific binding sites upon the gp140 Env protein, a competition ELISA was carried out with human MAbs obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: 2G12 and 2F5 (both provided by H. Katinger), 447-52D (provided by S. Zolla-Pazner), VRC01 (provided by John Mascola), and b12 (provided by D. Burton and C. Barbas). Briefly, 96-well polystyrene plates were coated with gp140 (AD8), washed as described above, and then blocked for 1 h with 5% skim milk powder in PBST. A total of 1 μg/μl of purified HBC IgG or a 1:100 dilution of whole HBC diluted in 5% skim milk powder in PBST was added and incubated for 2 h and washed as described above before serial dilutions of the MAbs were added. After 2 h of incubation, wells were washed and binding of MAbs to gp140 was detected using HRP-conjugated goat anti-human IgG antibody (Dako). Color reactions were developed using TMB substrate and stopped with 1 M H₂SO₄. Absorbance was measured at 450 nm against a reference of 690 nm.

SDS-PAGE and Western blotting.

Protein samples were resolved by 8% reducing SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane, blocked with 5% casein in PBS overnight at 4°C, and probed with sera or HBC for 4 h at room temperature. Bound IgG was detected using HRP-conjugated rabbit anti-bovine IgG antibody (Sigma) and Supersignal West Pico chemiluminescent substrate (Thermo Scientific). Bands were visualized using Kodak 4000MM Image Station and IM software (Carestream Health). As a loading control, all blots were stripped using RestoreTM Plus Western blot stripping buffer (Thermo Scientific) according to the manufacturer's protocol, washed with PBS and PBST, and blocked in 5% skim milk powder in PBST before being reprobed with pooled human HIV-positive serum (clade B) for 2 h. Bound human IgG was detected using goat anti-human IgG HRP-conjugated antibody (Invitrogen).

Production of EGFP Env-pseudotyped HIV-1 viruses.

Env-pseudotyped HIV-1 particles were produced by cotransfecting HeLa cells with an EGFP-expressing proviral reporter plasmid that terminates Env after 26 amino acids (pNL-4.3ΔenvNef-EGFP) (1) and a vector expressing the full-length Env protein of the strain of interest. The indicated HIV-1 Env-encoding plasmids were used to produce EGFP Env-pseudotyped viruses: AD8 (pCMV-AD8; prepared from pAD8 provided by M. Martin); MN (pSVIII-MN; provided by J. Sodroski); SF162 (pCAGGS-SF162; provided by L. Stamatatos and C. Cheng-Mayer); NL4.3 (pCMV-NL4.3; prepared from pNL4.3 provided by M. Martin); 89.6 (pSVIII-89.6; J. Sodroski); 966 (pCRII-93TH966-89; provided by B. H. Hahn); plasmids from the clade B and C NIH reference panel (AIDS Research and Reference Reagent Program); Du156.12 and Du172.17 (D. Montefiori, F. Gao, S. Abdool Karim, and G. Ramjee.); ZM197M.PB7, ZM214M.PL15, ZM233M.PB6, and ZM249M.PL1 (B. H. Hahn, Y. Li, and J. F. Salazar-Gonzalez); ZM53M.PB12, ZM109F.PB4, and ZM135M.PL10a (E. Hunter and C. Derdeyn); CAP45.2.00.G3 and CAP210.2.00E8 (L. Morris, K. Mlisana, and D. Montefiori); 6535, clone 3, PVO, clone 4, QH0692, clone 42, TRO, clone 11, AC10.0, clone 29, and SC 422661.8 (B. H. Hahn and J. F. Salazar-Gonzalez); and pWITO4160 clone 33, pTRJO4551 clone 58, pREJO4541 clone 67, and pRHPA4259 clone 7 (B. H. Hahn, X. Wei, and G. M. Shaw).

EGFP neutralization assay.

The neutralization assay was performed as previously described (11). Briefly, EGFP Env-pseudotyped viruses were incubated in a total volume of 30 μl for 1 h at 37°C with various concentrations of HBC (defatted and pasteurized) or purified HBC IgG. CF2th/CD4/CCR5/CXCR4 canine cells (17, 18) that express only the stable-transfected human receptors CD4, CCR5, and CXCR4 on the cell surface were used as target cells to exclude other human receptors that might complicate the analysis from any antibody reactive to any possible human contaminants in the gp140 vaccine. After incubation of virus and Ab, CF2th/CD4/CCR5/CXCR4 cells (2 × 10⁴/well) were added followed by spinoculation (42) at 1,200 × g for 2 h at room temperature. Residual virus and antibody were removed, and 200 μl of fresh medium was added to the cells, which were cultured for an additional 2 days. Target cells were analyzed for infection (positive for EGFP expression) by flow cytometry (FACSort; Becton, Dickinson). The percent neutralization was calculated as [1 − (virus + HBC or purified HBC IgG/virus + media)] × 100 and presented as the mean from duplicate samples. Our assay was previously validated with broadly NAbs 2F5, 2G12, and 4E10, giving a maximum of 75% neutralization at a concentration of 0.5 μg/μl (data not shown).

RESULTS

Vaccination elicits high IgG binding titers to gp140 in serum and colostrum.

All vaccinated cows seroconverted 2 weeks after the first boost vaccination (week nine), with mean anti-AD8 gp140 (clade B) IgG reciprocal serum endpoint titers of 1 × 10^2.75 (P-B), 1 × 10^1.5 (P-trimix), 1 × 10^4.5 (NP-B), and 1 × 10^4.75 (NP-trimix) (Fig. 1B). A second boost vaccination (week 12) did not lead to an increase in serum reciprocal IgG endpoint titers. Overall, serum IgG specific to clade B (AD8) was detectable in all cows but was highest for NP-B and NP-trimix. Serum IgG specific to clade A (UG8) and clade C (MW) was detected only in the NP group of cows and did not require vaccination with the trimix-clade vaccine (Fig. 1C), suggesting reactivities to conserved epitopes.

Three out of four cows had readily detectable anti-AD8 gp140 IgG titers of 1 × 10^3.5 (P-trimix), 1 × 10⁵ (NP-B), and 1 × 10^4.5 (NP-trimix) in HBC (Fig. 1D). In agreement with serum titers and breadth of reactivity, NP-B and NP-trimix HBC IgG endpoint titers against clade A and clade C gp140 were up to 1 × 10^4.75 and 1 × 10^3.5, respectively. The gp140-specific IgG titer of HBC from P-B (1 × 10²) was very low and therefore was not included in further characterizations.

Total IgG was purified from HBC to an overall purity of ∼90% (data not shown) and tested for binding to gp140. Purified HBC IgG from P-trimix showed weak binding to clade A, B, and C gp140 (Fig. 1E). In contrast, purified HBC IgG from NP-B and NP-trimix showed strong and equivalent reactivities to gp140 of all three clades, resulting in low endpoint concentrations between 0.78 μg/μl and 6.26 μg/μl. No binding was detected for a nonimmune BC IgG control.

Serum and colostrum IgG are specific to gp140 of clade A, B, and C.

Serum and HBC from both NP cows demonstrated strong and specific binding to secreted clade A, clade B, and clade C gp140 by immunoblotting (Fig. 2). No specific IgG responses were raised to proteins of the producer cell line (mock-transfected cells and empty vector pN1 control). No gp140 reactivity was observed for serum and HBC at the concentrations tested from animal P-trimix or prevaccination sera from all cows (data not shown). Nonimmune BC also had no gp140 reactivity (see Fig. S1 in the supplemental material).

Fig 2.

Binding specificity of antibodies determined by Western blotting. Specificity of serum (A and B) and colostrum (C and D) IgG of NP cows against secreted gp140 clade A (UG8), B (AD8), and C (MW) Env (black arrows). Cell culture supernatant from mock-transfected cells and cells transfected with vector backbone pN1 was used as a control. Proteins were separated on an 8% SDS gel under reducing conditions, and transferred proteins were blotted against a serum dilution of 1:100 or a colostrum dilution of 1:10,000 and rabbit anti-bovine IgG HRP-conjugated antibody. Efficient loading of Env on the membranes was assessed by reprobing with pooled HIV-positive sera (clade B) (1:5,000) and goat anti-human IgG HRP-conjugated antibody (1:1,000).

HBC mediates broad neutralization of HIV-1-pseudotyped viruses.

We assessed HIV-1 neutralization breadth of one nonimmune BC and three HBCs using a panel of 27 Env-pseudotyped HIV-1 viruses with a broad range of neutralization sensitivities from clades B (n = 15), C (n = 11), and the circulating recombinant form (CRF) A/E (n = 1). In the neutralization assay used, typically 30% to 80% neutralization is seen using sensitive Env strains and patient-derived sera at a similar dilution range (data not shown). Therefore, neutralization was divided in 3 categories: absent/weak (0 to 33%), moderate (33 to 66%), and strong (>66%) neutralization. A summary of the neutralization profiles of HBC is given in Table 1. We found that nonimmune BC showed intrinsic neutralization with a baseline activity between 13.7% and 64%. This neutralizing activity may result from high concentrations of low-affinity antibodies against Env that could dissociate in other assays (e.g., ELISA) that use lower concentrations of antibodies. Surprisingly HBC from NP-trimix, with the highest gp140 IgG binding titer, was less potent than HBC from animal P-trimix or NP-B which showed strong to moderate neutralization for almost all tested viruses ranging from 33.7 to 89.7% (P-trimix) and 42.8 to 94.6% (NP-B) neutralization. HBC from NP-B not only had the most potent neutralization activity but also exhibited strong breadth, neutralizing 1/1 CRF AE and 15/15 clade B and 9/11 clade C strains. Thus, 2 of 3 HBC samples showed broad neutralizing activity against sensitive to moderately resistant HIV-1 Env clones from different clades. Total IgG concentrations in HBC, however, varied from 57.7 μg/μl (P-trimix), 60 μg/μl (NP-B), 26.6 μg/μl (NP-trimix), and 20 μg/μl (nonimmune), and this may in part explain the different neutralization profiles.

Table 1.

HIV-1 neutralization profile of whole HBC

^aReduction of EGFP-expressing CF2³ canine thymocyte cells after infection with Env-pseudotyped viruses preincubated with defatted and pasteurized colostrum was detected in duplicate wells. Numbers represent percent neutralization for a 1:16 dilution against the indicated Env-pseudotyped viruses, including common lab strains and the NIH clade B and C reference panels. The percentage of neutralization was calculated as follows: [1 − (virus + colostrum or antibody/virus + media)] × 100. Neutralization is presented as the means ± SD from duplicate samples. Results are shown for one representative experiment of 3 repeats.

^bEGFP Env-pseudotyped virus with respective envelope protein.

^cNeutralization sensitivity for NIH reference Env clones are given according to Li et al. (28, 29) and include genetic and antigenic diversity while avoiding strains that are unusually sensitive or resistant to neutralization. Pseudovirus sensitivities are defined as follows: sensitive, readily neutralized by the majority of patient serum and monoclonal antibodies; moderately sensitive/resistant, neutralized by a small proportion of patient serum and monoclonal antibodies; resistant, neutralized by very few patient serum and monoclonal antibodies.

Purified HBC IgG retains broad neutralizing activity.

As BC contains different concentrations of IgG and intrinsic antibacterial and antiviral proteins and peptides, we investigated if broad HIV-1-neutralizing activity was also mediated by IgG. Neutralizing activity of purified HBC IgG (0.01 to 1 μg/μl) was tested against a representative panel of Env-pseudotyped HIV-1 strains, including 1 clade CRF AE strain, 5 common lab strains of clade B, and 2 clade B and 2 clade C NIH reference Env strains described above.

Purified HBC IgG from NP-B showed the highest activity with strong to moderate neutralization observed for all viruses tested (n = 10) at IgG concentrations of 1 to 0.1 μg/μl (Fig. 3A). IgG from NP-trimix also displayed strong to moderate neutralization for all viruses at concentrations of 1 μg/μl and 0.5 μg/μl and 5/10 viruses at concentrations of 0.1 μg/μl. IgG from P-trimix was less potent and showed moderate neutralization for 10/10 and 8/10 viruses at a concentration of 1 μg/μl and 0.5 μg/μl, respectively. Neutralization was weak or absent at IgG concentrations of less than 0.1 μg/μl except for NP-B, which displayed moderate neutralization at this concentration. No viral neutralization was observed using equivalent concentrations of control IgG purified from human or bovine sera (Sigma). The breadth of neutralization for each IgG sample at a concentration of 0.5 μg/μl is shown in Fig. 3B.

Fig 3.

Neutralization profile of purified colostrum IgG against a representative panel of Env-pseudotyped viruses with different neutralization sensitivities. Colostrum purified IgG was used at 0.01 to 1 μg/μl. (A) Average neutralization of all viruses tested plotted against the respective purified colostrum IgG concentration. IgGs purified from human and bovine HIV-negative serum (Sigma) were included as controls for nonspecific neutralization. (B) Representative neutralizing profile for all IgG samples against the 10 viruses for an IgG concentration of 0.5 μg/μl. NI_max and NI_min represent a strong neutralizing and nonneutralizing nonimmune colostrum sample, respectively (out of 6 viruses tested). Dotted lines indicate the moderate and strong neutralization cutoffs and 0% neutralization. Results are expressed as mean percentages of neutralization from two independent experiments; percentage of neutralization was calculated as [1 − (virus + colostrum or antibody/virus + media)] × 100. Neutralization is shown as means from duplicate samples for one representative experiment of 3 repeats.

We tested BC-purified IgG from an increased number (n = 6) of HIV-1 Env naïve animals and detected a wide range of intrinsic neutralization potency. Figure 3B shows the highest- and lowest-neutralizing samples of nonimmune BC IgG at concentrations of 0.5 μg/μl. Measured neutralizing activity of these BC-purified IgGs were similar to the neutralizing activity of corresponding whole BC samples (see Fig. S2 in the supplemental material).

Purified HBC IgG shows activity against the CD4 binding site of Env.

To define potential neutralizing sites of Env targeted by colostrum IgG from NP-B and NP-trimix, we performed competition ELISAs with human MAbs 2G12, 2F5, 44752D, VRC01, and b12. Purified HBC IgG from NP-B and NP-trimix specifically inhibited the binding of b12 and VRC01 to gp140 with similar potencies but did not inhibit binding of MAb 2G12 (Fig. 4) or 2F5 or 44752D (data not shown). This b12/VRC01-blocking activity was not observed using high concentrations of IgG from the nonimmune control, suggesting that vaccination of cows with gp140 can induce Abs which target the CD4 binding site of Env. Competition with MAb b12 and VRC01 binding was not observed for the same concentrations of purified HBC IgG from P-trimix (data not shown).

Fig 4.

Mapping of Env binding epitopes. Colostrum IgG competes with human neutralizing MAb b12 and VRC01 for binding at the gp140 CD4 binding site. (A) Cartoon of an HIV gp140 envelope protein (V2/3 loop in red, V3 loop in gray, and gp41 in orange) with the respected binding sites of the monoclonal antibodies used. Competition ELISAs were performed by titrating the human neutralizing MAb b12, VRC01, and 2G12 against a constant background of 100 μg purified colostrum IgG (B to E) or a 1:100 dilution of whole hyperimmune colostrum (F). The ability of b12, VRC01, and 2G12 to bind to gp140 Env (AD8) in the presence or absence of colostrum IgG was detected by anti-human IgG HRP-conjugated antibody.

DISCUSSION

A vaccination or immunotherapy strategy inducing broadly neutralizing Abs capable of preventing transmission of HIV-1 is considered the best method of curtailing the increasing costs of HIV-1 treatment; however, such a vaccine has not yet been produced. As an alternative, we aimed to produce broadly neutralizing anti-HIV Abs at a sufficiently low cost that they could be coformulated into a topical microbicide as an HIV prevention strategy. Here, we successfully induced high titers of gp140-specific polyclonal Abs with broad HIV-1-neutralizing activity in HBC by vaccinating pregnant cows with purified gp140 oligomers.

Vaccination with gp140 from a single clade (AD8) was sufficient to induce IgG with robust cross-clade binding and neutralization. We did not observe any increased neutralization breadth by vaccination with multiclade gp140 antigens. In contrast with other studies (2, 11, 19, 53), we found broad neutralizing activity from HBC and more importantly from purified HBC IgG in a pseudovirus-based assay that included tier 1 and tier 2 Env and primary isolates from heterologous clade viral isolates. The breadth of neutralization seen with our purified HBC IgG, including primary Env clones derived from sexually acquired acute/early infections, demonstrates clear potential for inclusion of HBC-derived polyclonal antibodies in candidate microbicides aiming to prevent sexually acquired HIV.

The highest serum and HBC titers of gp140-specific Abs with broad neutralizing activity were induced in cows vaccinated before conception (NP). The extended duration and one extra boost of the NP vaccination protocol may favor IgG affinity maturation that has been associated with effective HIV neutralization (64). Further improvements in the magnitude of vaccine-elicited immune responses might be achieved by optimization of the dose and adjuvant used with the gp140 immunogens. Previous immunogenicity studies of gp140 trimers in mice, rabbits, guinea pigs, and primates have used protein concentrations ranging from 5 μg and 500 μg (2, 6, 11, 23, 41). Considering body weight, vaccination in the current study with only 100 μg of gp140 may be suboptimal but still produced high levels of neutralizing antibodies. Although the number of animals used in this study is limited, results suggest that for future vaccination studies, vaccination should be carried out prior to conception followed by multiple boost vaccinations during pregnancy with gp140 oligomer. We found that AD8 gp140 alone was sufficient. AD8 expresses well in cell culture, allowing for production of sufficient amounts necessary for vaccination. Abundant antigen is important, as vaccinating with an increased amount of gp140 in conjunction with at least 10 cows may be a strategy to reduce variability and improve consistency. Another effective way to reduce biological variability is to screen the colostrum for each animal prior to pooling or in large-scale manufacture to have at least 100 cows per production run. This is a standard method of Immuron Ltd. to achieve consistency for the production of their oral colostrum IgG product to prevent traveler's diarrhea (Travelan). In that case, non- or low responders to vaccination would have a limited impact on the final product.

Polyclonal IgG, including the HBC IgG from this study, have specificity for many different epitopes and generally contain only a proportion of IgG specific to the vaccine antigen, such as gp140 in our case. Therefore, a relatively small proportion of HBC IgG is likely to be directly involved in neutralization. This explains why the HBC IgG here typically had lower potency than the previously characterized human broadly mNAbs, despite its broad neutralizing activity. Protocols that further refine the vaccination for polyclonal HIV-neutralizing IgG may improve this yield. Nevertheless, substantial amounts of antibody could be prepared with the described protocols by harnessing commercial dairies that routinely process milk products for human consumption. This vast protein production system could produce unprecedented quantities of NAb for mucosal applications at a relatively low cost.

We observed a relationship between IgG concentration and HIV neutralization from whole HBC and purified HBC IgG. For neutralization measurements of purified HBC IgG, IgG concentrations were equalized to better reflect the IgG-mediated neutralizing activity. The presence and concentrations of antimicrobial components within BC, including lactoferrin, lysozyme, or lactoperoxidase, vary considerably among healthy cows, are highly dependent on the exact timing of BC collection, and could therefore significantly influence the neutralization profile of HBC (12, 15, 38, 55). We mapped the Ab-binding sites within gp140 conferring the neutralizing activities in purified HBC IgG from both NP cows using competition with MAbs with defined epitopes. Purified HBC IgG was able to block the binding of the MAb b12 and VRC01, which target the CD4 binding site of Env and prevents CD4 attachment (65). The functionally conserved CD4 binding region of gp120 is a crucial Ab epitope and has previously been linked with exceptionally broad neutralization (63). Moreover, defined neutralizing activity within the plasma of HIV-infected patients is most commonly directed toward to the CD4 binding site (4, 30). Although binding to the CD4 binding site does not necessarily guarantee broad neutralization, our b12 blocking and VRC01 data demonstrates binding of purified HBC IgG to the CD4 binding site, which may explain, at least in part, the broad neutralizing activity of purified HBC IgG. Molecular characterization of b12 and several other patient-derived broadly mNAbs has revealed that these Abs have an unusually long complementarity-determining region (CDR) of the antibody heavy chain 3 (H3) (18 residues and longer) that is vital for Ab-neutralizing activity (7, 9, 22, 43, 48, 49, 54, 61, 66, 67). Extended CDR H3 regions represent an important alteration of Ab structure that facilitates the insertion of binding domains into recessed cavities within gp120. The bovine heavy chain repertoire utilizes Ig VH genes from only a single family, and significant Ab diversity is generated by extensive somatic mutation (3, 31). The bovine CDR H3 is particularly variable and ranges in length from 13 to 28 amino acids, with the majority of Abs containing a CDR H3 in excess of 21 amino acids (50). This is longer than has been reported for humans (62) and may provide one potential explanation for the exceptional neutralization breadth of HBC IgG.

Our results demonstrate that vaccination of cows with purified gp140 oligomers is an effective strategy for the production of high concentrations of HIV-1-specific Abs with gp140 binding and broad neutralizing activity. Although Abs can be produced using recombinant biological manufacturing techniques, anti-HIV IgG from HBC represents an inexpensive source of very large quantities of Abs using established dairy manufacturing protocols that are potentially transferrable to developing countries.

Further clinical development of the HIV-1-specific HBC IgG will require thorough confirmation of the neutralizing activity by an external reference laboratory with standardized virus production and assay conditions and different cells, as it was recently shown that neutralizing activity of polyclonal Ab preparations if used to prevent HIV infection is essential (44). In addition, in vivo experiments confirming the safety and antiviral potential of HIV-1-specific HBC IgG are warranted. The ultimate goal would be to combine HIV-1-specific IgG with other suitable components, such as antiretroviral drugs, or compounds that are currently under evaluation in topical microbicide formulations in human clinical trials.