Neutralizing antibodies induced in immunized macaques recognize the CD4-binding site on an occluded-open HIV-1 envelope trimer.

Broadly-neutralizing antibodies (bNAbs) against HIV-1 Env can protect from infection. We characterize Ab1303 and Ab1573, heterologously-neutralizing CD4-binding site (CD4bs) antibodies, isolated from sequentially-immunized macaques. Ab1303/Ab1573 binding is observed only when Env trimers are not constrained in the closed, prefusion conformation. Fab-Env cryo-EM structures show that both antibodies recognize the CD4bs on Env trimer with an 'occluded-open' conformation between closed, as targeted by bNAbs, and fully-open, as recognized by CD4. The occluded-open Env trimer conformation includes outwardly-rotated gp120 subunits, but unlike CD4-bound Envs, does not exhibit V1V2 displacement, 4-stranded gp120 bridging sheet, or co-receptor binding site exposure. Inter-protomer distances within trimers measured by double electron-electron resonance spectroscopy suggest an equilibrium between occluded-open and closed Env conformations, consistent with Ab1303/Ab1573 binding stabilizing an existing conformation. Studies of Ab1303/Ab1573 demonstrate that CD4bs neutralizing antibodies that bind open Env trimers can be raised by immunization, thereby informing immunogen design and antibody therapeutic efforts.

SOSIP Trimer-Specific Antibodies Isolated from a Simian-Human Immunodeficiency Virus-Infected Monkey with versus without a Pre-blocking Step with gp41.

BG505 SOSIP.664 (hereafter referred to as SOSIP), a stabilized trimeric mimic of the HIV-1 envelope spike resembling the native viral spike, is a useful tool for isolating anti-HIV-1 neutralizing antibodies. We screened long-term SHIV-AD8 infected rhesus monkeys for potency and breadth of serum neutralizing activity against autologous and heterologous viruses: SHIV-AD8, HIV-1 YU2, HIV-1 JR-CSF, and HIV-1 NL4-3. Monkey rh2436 neutralized all viruses tested and showed strong reactivity to the SOSIP trimer, suggesting this was a promising candidate for attempts at monoclonal antibody (MAb) isolation. MAbs were isolated by performing single B-cell sorts from peripheral blood mononuclear cells (PBMC) by FACS using the SOSIP trimer as a probe. An initial round of sorted cells revealed the majority of isolated MAbs were directed to the gp41 external domain portion of the SOSIP trimer and were mostly non-neutralizing against tested isolates. A second sort was performed, introducing a gp41 blocking step prior to PBMC staining and FACS sorting. These isolated MAbs bound SOSIP trimer but were no longer directed to the gp41 external domain portion. A significantly higher proportion of MAbs with neutralizing activity were obtained with this strategy. Our data show this pre-blocking step with gp41 greatly increases the yield of non-gp41-reactive, SOSIP-specific MAbs and increases the likelihood of isolating MAbs with neutralizing activity. IMPORTANCE Recent advancements in the field have focused on the isolation and use of broadly neutralizing antibodies for both prophylaxis and therapy. Finding a useful probe to isolate broad potent neutralizing antibodies while avoiding non-neutralizing antibodies is important. The SOSIP trimer has been shown to be a great tool for this purpose because it binds known broadly neutralizing antibodies. However, the SOSIP trimer can isolate non-neutralizing antibodies as well, including gp41-specific MAbs. Introducing a pre-blocking step with gp41 recombinant protein decreased the percent of gp41-specific antibodies isolated with SOSIP probe, as well as increased the number of neutralizing antibodies isolated. This method can be used as a tool to increase the chances of isolating neutralizing antibodies.

Characterisation of a highly potent and near pan-neutralising anti-HIV monoclonal antibody expressed in tobacco plants.

BACKGROUND: HIV remains one of the most important health issues worldwide, with almost 40 million people living with HIV. Although patients develop antibodies against the virus, its high mutation rate allows evasion of immune responses. Some patients, however, produce antibodies that are able to bind to, and neutralise different strains of HIV. One such 'broadly neutralising' antibody is 'N6'. Identified in 2016, N6 can neutralise 98% of HIV-1 isolates with a median IC50 of 0.066 µg/mL. This neutralisation breadth makes N6 a very promising therapeutic candidate. RESULTS: N6 was expressed in a glycoengineered line of N. benthamiana plants (pN6) and compared to the mammalian cell-expressed equivalent (mN6). Expression at 49 mg/kg (fresh leaf tissue) was achieved in plants, although extraction and purification are more challenging than for most plant-expressed antibodies. N-glycoanalysis demonstrated the absence of xylosylation and a reduction in α(1,3)-fucosylation that are typically found in plant glycoproteins. The N6 light chain contains a potential N-glycosylation site, which was modified and displayed more α(1,3)-fucose than the heavy chain. The binding kinetics of pN6 and mN6, measured by surface plasmon resonance, were similar for HIV gp120. pN6 had a tenfold higher affinity for FcγRIIIa, which was reflected in an antibody-dependent cellular cytotoxicity assay, where pN6 induced a more potent response from effector cells than that of mN6. pN6 demonstrated the same potency and breadth of neutralisation as mN6, against a panel of HIV strains. CONCLUSIONS: The successful expression of N6 in tobacco supports the prospect of developing a low-cost, low-tech production platform for a monoclonal antibody cocktail to control HIV in low-to middle income countries.

Autologous IgG antibodies block outgrowth of a substantial but variable fraction of viruses in the latent reservoir for HIV-1.

In untreated HIV-1 infection, rapid viral evolution allows escape from immune responses. Viral replication can be blocked by antiretroviral therapy. However, HIV-1 persists in a latent reservoir in resting CD4+ T cells, and rebound viremia occurs following treatment interruption. The reservoir, which is maintained in part by clonal expansion, can be measured using quantitative viral outgrowth assays (QVOAs) in which latency is reversed with T cell activation to allow viral outgrowth. Recent studies have shown that viruses detected in QVOAs prior to treatment interruption often differ from rebound viruses. We hypothesized that autologous neutralizing antibodies directed at the HIV-1 envelope (Env) protein might block outgrowth of some reservoir viruses. We modified the QVOA to reflect pressure from low concentrations of autologous antibodies and showed that outgrowth of a substantial but variable fraction of reservoir viruses is blocked by autologous contemporaneous immunoglobulin G (IgG). A reduction in outgrowth of >80% was seen in 6 of 15 individuals. This effect was due to direct neutralization. We established a phylogenetic relationship between rebound viruses and viruses growing out in vitro in the presence of autologous antibodies. Some large infected cell clones detected by QVOA carried neutralization-sensitive viruses, providing a cogent explanation for differences between rebound virus and viruses detected in standard QVOAs. Measurement of the frequency of reservoir viruses capable of outgrowth in the presence of autologous IgG might allow more accurate prediction of time to viral rebound. Ultimately, therapeutic immunization targeting the subset of variants resistant to autologous IgG might contribute to a functional cure.

Feasibility and acceptability of a pilot, peer-led HIV self-testing intervention in a hyperendemic fishing community in rural Uganda.

BACKGROUND: Novel interventions are needed to reach young people and adult men with HIV services given the low HIV testing rates in these population sub-groups. We assessed the feasibility and acceptability of a peer-led oral HIV self-testing (HIVST) intervention in Kasensero, a hyperendemic fishing community (HIV prevalence: 37-41%) in Rakai, Uganda. METHODS: This study was conducted among young people (15-24 years) and adult men (25+ years) between May and August 2019. The study entailed distribution of HIVST kits by trained "peer-leaders," who were selected from existing social networks and trained in HIVST distribution processes. Peer-leaders received up to 10 kits to distribute to eligible social network members (i.e. aged 15-24 years if young people or 25+ years if adult man, not tested in the past 3 months, and HIV-negative or of unknown HIV status at enrolment). The intervention was evaluated against the feasibility benchmark of 70% of peer-leaders distributing up to 70% of the kits that they received; and the acceptability benchmark of >80% of the respondents self-testing for HIV. RESULTS: Of 298 enrolled into the study at baseline, 56.4% (n = 168) were young people (15-24 years) and 43.6% (n = 130) were adult males (25+ years). Peer-leaders received 298 kits and distributed 296 (99.3%) kits to their social network members. Of the 282 interviewed at follow-up, 98.2% (n = 277) reported that they used the HIVST kits. HIV prevalence was 7.4% (n = 21). Of the 57.1% (n = 12) first-time HIV-positives, 100% sought confirmatory HIV testing and nine of the ten (90%) respondents who were confirmed as HIV-positive were linked to HIV care within 1 week of HIV diagnosis. CONCLUSION: Our findings show that a social network-based, peer-led HIVST intervention in a hyperendemic fishing community is highly feasible and acceptable, and achieves high linkage to HIV care among newly diagnosed HIV-positive individuals.

Isolation of single HIV-1 Envelope specific B cells and antibody cloning from immunized rhesus macaques.

Antibody cloning from single B cells is an essential tool for characterizing humoral immune responses and obtaining valuable therapeutic and analytical reagents. Antibody cloning from individuals with high serologic titers to HIV-1, Influenza, Malaria and ZIKV has led to new insights that inform vaccine design efforts. In contrast to humans and mice, less is known about antibody cloning from single B cells in macaques. Here, we describe a protocol to identify and purify single antigen-specific macaque B cells, and subsequently clone and produce macaque monoclonal antibodies. The sorting strategy requires the use of a combination of fluorochrome labeled antigens and omission of anti-IgG antibodies that can interfere with antigen binding and vice versa. Optimized methods for macaque antibody gene amplification, DNA preparation for antibody production and antibody screening by ELISA are also presented.

Broad and Potent Neutralizing Antibodies Recognize the Silent Face of the HIV Envelope.

Broadly neutralizing antibodies (bNAbs) against HIV-1 envelope (Env) inform vaccine design and are potential therapeutic agents. We identified SF12 and related bNAbs with up to 62% neutralization breadth from an HIV-infected donor. SF12 recognized a glycan-dominated epitope on Env's silent face and was potent against clade AE viruses, which are poorly covered by V3-glycan bNAbs. A 3.3Å cryo-EM structure of a SF12-Env trimer complex showed additional contacts to Env protein residues by SF12 compared with VRC-PG05, the only other known donor-derived silentface antibody, explaining SF12's increased neutralization breadth, potency, and resistance to Env mutation routes. Asymmetric binding of SF12 was associated with distinct N-glycan conformations across Env protomers, demonstrating intra-Env glycan heterogeneity. Administrating SF12 to HIV-1-infected humanized mice suppressed viremia and selected for viruses lacking the N448gp120 glycan. Effective bNAbs can therefore be raised against HIV-1 Env's silent face, suggesting their potential for HIV-1 prevention, therapy, and vaccine development.

Kappa chain maturation helps drive rapid development of an infant HIV-1 broadly neutralizing antibody lineage.

HIV-infected infants develop broadly neutralizing plasma responses with more rapid kinetics than adults, suggesting the ontogeny of infant responses could better inform a path to achievable vaccine targets. Here we reconstruct the developmental lineage of BF520.1, an infant-derived HIV-specific broadly neutralizing antibody (bnAb), using computational methods developed specifically for this purpose. We find that the BF520.1 inferred naive precursor binds HIV Env. We also show that heterologous cross-clade neutralizing activity evolved in the infant within six months of infection and that, ultimately, only 2% SHM is needed to achieve the full breadth of the mature antibody. Mutagenesis and structural analyses reveal that, for this infant bnAb, substitutions in the kappa chain were critical for activity, particularly in CDRL1. Overall, the developmental pathway of this infant antibody includes features distinct from adult antibodies, including several that may be amenable to better vaccine responses.

An innovative vending machine-based HIV testing and intervention service in China: anonymous urine collection kits distributed at universities.

To find more effective test and intervention measures, and to achieve the first 90 of the 90-90-90 target, this study was conducted for the first time to develop and assess an innovative HIV anonymous urine test service-based vending machine and Internet at universities of China. From June to December 2016, 11 vending machines were placed in 7 pilot universities in Beijing, Sichuan, Yunnan and Heilongjiang provinces. A total of 957 HIV urine collection kits were dispensed free and also through vending machines and 378 (39.5%) urine samples were returned and 376 (99.5%) of them were qualified to be tested for HIV antibody in professional laboratories. Participants searched for confidential test results using an ID code online. Only seven (1.86%) urine samples were positive. Monitoring data showed 67.8% (255/376) participants searched for test results online, 72.2% of kits were purchased in dormitory buildings and 27.8% were purchased in teaching buildings and 88.9% were purchased between 21:00 and 24:00. In conclusion, this study analyzes the acceptability, feasibility and effectiveness of HIV testing and intervention service.

Phagocytosis of a Model Human Immunodeficiency Virus Target by Human Breast Milk Leukocytes Is Predominantly Granulocyte-Driven When Elicited by Specific Antibody.

BACKGROUND: Studies demonstrate a protective effect of antibodies (Abs) in breast milk (BM) against mother-to-child transmission (MTCT) of human immunodeficiency virus (HIV). Contribution of the BM cellular component has been overlooked. The only clinical HIV vaccine trial to demonstrate efficacy, RV144, correlated protection with Abs mediating functions through the constant immunoglobulin region-the crystallizable fragment (Fc). These data support induction of vaccine Abs triggering antiviral activities by leukocytes through Fc receptors (FcRs). OBJECTIVE: To measure Ab-dependent cellular phagocytosis (ADCP), an essential Fc-mediated response, by BM phagocytes. MATERIALS AND METHODS: Cells were isolated from five human BM samples obtained at 7-183 days postpartum and analyzed for ADCP. Fluorescent beads coated with HIV envelope (Env) epitopes were used as targets. Sixty-seven to 100 mL of milk was utilized. RESULTS: Total cell concentrations per milliliter were 16,083-222,857, with 1.6-12.3% being CD45+ leukocytes. ADCP activity was measurable using the HIV-specific Ab 830A. Use of the actin inhibitor cytochalasin D and FcR blocker indicated that ADCP was actin dependent and required FcR engagement. ADCP scores were variable, but largely consistent, across the samples studied, exhibiting <4-fold difference from lowest to highest activity for CD45+ cells. Of the CD45+ ADCP, significantly more activity was granulocyte derived (72-95%), while the remaining activity was monocyte driven. CONCLUSIONS: The data indicate that BM phagocytes can manifest antiviral activities in the presence of specific Abs and therefore may contribute to reduction of MTCT of HIV.

Increased breadth of HIV-1 neutralization achieved by diverse antibody clones each with limited neutralization breadth.

Broadly neutralizing antibodies (bNAbs) are rarely elicited by current human immunodeficiency virus type 1 (HIV-1) vaccine designs, but the presence of bNAbs in naturally infected individuals may be associated with high plasma viral loads, suggesting that the magnitude, duration, and diversity of viral exposure may contribute to the development of bNAbs. Here, we report the isolation and characterization of a panel of human monoclonal antibodies (mAbs) from two subjects who developed broadly neutralizing autologous antibody responses during HIV-1 infection. In both subjects, we identified collections of mAbs that exhibited specificity only to a few autologous envelopes (Envs), with some mAbs exhibiting specificity only to a subset of Envs within the quasispecies of a particular sample at one time point. Neutralizing antibodies (NAbs) isolated from these subjects mapped mostly to epitopes in the Env V3 loop region and the CD4 binding site. None of the individual neutralizing mAbs recovered exhibited the cumulative breadth of neutralization present in the serum of the subjects. Surprisingly, however, the activity of polyclonal mixtures comprising individual mAbs that each possessed limited neutralizing activity, could achieve increased breadth of neutralizing activity against autologous isolates. While a single broadly neutralizing antibody targeting one epitope can mediate neutralization breadth, the findings presented here suggest that a cooperative polyclonal process mediated by diverse antibodies with more limited breadth targeting multiple epitopes also can achieve neutralization breadth against HIV-1.

HIV screening among patients seeking care at Xuanwu Hospital: A cross-sectional study in Beijing, China, 2011-2016.

OBJECTIVES: One-third of people living with HIV in China are still unaware of their status, so we sought to better understand HIV testing in the general hospital setting in China. METHODS: A cross-sectional study was conducted using the electronic medical records of all patients who attended Xuanwu Hospital in Beijing, January 1, 2011 to December 31, 2016. HIV screening and detection rates and characteristics of patients diagnosed with HIV were assessed. RESULTS: Overall, 235,961 patients were screened, for a screening rate of 1.4%. Although most were outpatients (98.4%), screening rate was higher among inpatients (70.0% versus 0.4%), and highest in internal medicine (36.1%) and surgery (33.3%) departments. A total of 140 patients were diagnosed with HIV, for a detection rate of 5.93 per 10,000. Detection rates were highest among outpatients (9.34 per 10,000), and patients attending the dermatology and sexually transmitted infection (STI) department (153.85 per 10,000). Most diagnoses were made among males (91.4%), aged 20-39 (67.1%), who reported becoming infected through homosexual contact (70.0%). CONCLUSIONS: HIV screening in China's general hospitals needs to be improved. More focus should be placed on screening outpatients, especially in the dermatology and STI department, and young men.

Identification of a HIV Gp41-Specific Human Monoclonal Antibody With Potent Antibody-Dependent Cellular Cytotoxicity.

Antibody-Dependent Cellular Cytotoxicity (ADCC) is a major mechanism of protection against viral infections in vivo. Identification of HIV-1-specific monoclonal antibodies (mAbs) with potent ADCC activity may help develop an effective HIV-1 vaccine. In present study, we isolated such human mAb, designated E10, from an HIV-1-infected patient sample by single B cell sorting and single cell PCR. E10 bound to gp140 trimer and linear peptides derived from gp41 membrane proximal external region (MPER). E10 epitope (QEKNEQELLEL) overlapped with mAb 2F5 epitope. However, E10 differentiated from 2F5 in neutralization breadth and potency, as well as ADCC activity. E10 showed low neutralization activity and narrow spectrum of neutralization compared to 2F5, but it mediated higher ADCC activity than 2F5 at low antibody concentration. Fine mapping of E10 epitope may potentiate MPER-based subunit vaccine development.

V2-Directed Vaccine-like Antibodies from HIV-1 Infection Identify an Additional K169-Binding Light Chain Motif with Broad ADCC Activity.

Antibodies that bind residue K169 in the V2 region of the HIV-1 envelope correlated with reduced risk of infection in the RV144 vaccine trial but were restricted to two ED-motif-encoding light chain genes. Here, we identify an HIV-infected donor with high-titer V2 peptide-binding antibodies and isolate two antibody lineages (CAP228-16H/19F and CAP228-3D) that mediate potent antibody-dependent cell-mediated cytotoxicity (ADCC). Both lineages use the IGHV5-51 heavy chain germline gene, similar to the RV144 antibody CH58, but one lineage (CAP228-16H/19F) uses a light chain without the ED motif. A cocrystal structure of CAP228-16H bound to a V2 peptide identified a IGLV3-21 gene-encoded DDxD motif that is used to bind K169, with a mechanism that allows CAP228-16H to recognize more globally relevant V2 immunotypes. Overall, these data further our understanding of the development of cross-reactive, V2-binding, antiviral antibodies and effectively expand the human light chain repertoire able to respond to RV144-like immunogens.

Identification of a novel broadly HIV-1-neutralizing antibody from a CRF01_AE-infected Chinese donor.

The isolation and characterization of monoclonal broadly neutralizing antibodies (nAbs) from natural HIV-1-infected individuals play very important roles in understanding nAb responses to HIV-1 infection and designing vaccines and therapeutics. Many broadly nAbs have been isolated from individuals infected with HIV-1 clade A, B, C, etc., but, as an important recombinant virus, the identification of broadly nAbs in CRF01_AE-infected individuals remains elusive. In this study, we used antigen-specific single B-cell sorting and monoclonal antibody expression to isolate monoclonal antibodies from a CRF01_AE-infected Chinese donor (GX2016EU04), a broad neutralizer based on neutralizing activity against a cross-clade virus panel. We identified a series of HIV-1 monoclonal cross-reactive nAbs, termed F2, H6, BF8, F4, F8, BE7, and F6. F6 could neutralize 21 of 37 tested HIV-1 Env-pseudotyped viruses (57%) with a geometric mean value of 12.15 µg/ml. Heavy and light chains of F6 were derived from IGHV4-34 and IGKV 2-28 germlines, complementarity determining region (CDR) 3 loops were composed of 18 and 9 amino acids, and somatic hypermutations (SHMs) were 16.14% and 11.83% divergent from their respective germline genes. F6 was a GP120-specific nAb and recognized the linear epitope. We identified for the first time a novel broadly HIV-1-neutralizing antibody, termed F6, from a CRF01_AE-infected donor, which could enrich the research of HIV-1 nAbs and provide useful insights for designing vaccine immunogens and antibody-based therapeutics.

[Analysis on influencing factors that leading to nonspecific responses to indeterminate results of HIV antibodies].

Objective: To identify the influencing factors that leading to nonspecific responses to indeterminate HIV antibody tests, to provide scientific evidence for the differential diagnosis of HIV infection and control strategy. Methods: A case control study was conducted. The samples of HIV antibody indeterminate in confirmed Western blot (WB) tests, but were negative in HIV nucleic acid tests, were collected as HIV antibody indeterminate group from WB results of HIV confirmatory laboratories of Fujian province in 2015-2016. The general population matched group with HIV antibody screening negative samples and WB negative matched group with WB negative samples were selected as the two compared groups by matching gender and age from HIV antibody screening in Fujian province in the same period. Blood concentrations of hepatitis B surface antigen (HBsAg), anti-hepatitis C virus (HCV) antibody, anti-treponema pallidum (TP) antibody, antinuclear antibody (ANA), anti-human T-cell leukemia virus (HTLV) antibody, and alpha-fetoprotein (AFP) were detected by using enzyme-linked immunosorbent assay (ELISA). χ(2) test and multivariate non-conditional logistic regression analysis were performed to identify the influencing factors that leading to nonspecific responses, to indeterminate HIV antibody tests. Results: A total of 13 WB band patterns were observed in 110 HIV antibody indeterminate samples, in which a single p24 band (58.18%, 64/110), a single gp160 band (17.27%, 19/110) and a single p17 band (7.27%, 8/110) were the three most common patterns. The positive rate of anti-TP antibody was significantly higher in HIV antibody indeterminate samples than general population control group and WB negative control group (10.91%, 12/110 vs. 1.77%, 4/226 and 3.64%, 4/110), compared with two control groups (χ(2)=13.627 and 4.314, P<0.05). The positive rate of AFP was significantly higher in HIV antibody indeterminate samples than general population control group (18.18%, 20/110 vs. 0.44%, 1/226, χ(2)=39.736, P<0.05), the different was not significant compared with WB negative control group (18.18%, 20/110 vs. 23.64%, 26/110, χ(2)=0.990, P>0.05) While no significant differences were found between HIV antibody indeterminate group and two control groups in terms of the positive rates of ANA, HBsAg, anti-HCV antibody or anti-HTLV antibody. Conclusions: The influencing factors that leading to nonspecific responses to indeterminate HIV antibody tests appeared complicate, and the anti-TP antibody positivity might be an influencing factor responsible for nonspecific responses to indeterminate HIV antibody tests.

[Diagnostic and epidemiological features of the first two HIV-2 indigenous infections in Hunan province].

Objective: To study the diagnostic and epidemiological features of the first two HIV-2 indigenous cases in Hunan province. Methods: Blood samples from two individuals with "HIV antibody indeterminate" and HIV-2 specific band showed by HIV-1/2 western blotting method, were repeatedly collected and detected under HIV 1+2 strip immunoassay and PCR, in Changsha city, Hunan province, through March to November, 2017. An epidemiological survey was carried out at the same time. Results: Our findings showed that the two cases were sex partners, without histories of sexual contact with foreigners and the source of infection was unknown. Results from the HIV 1+2 antibody confirmation test showed that they were "HIV-2 antibody positive" . Through amplifying and sequencing the gag area of HIV-2 and BLAST, the similarity of HIV-2 strains presented as 98%. The results also showed that there were HIV-2 specific fragments in the two cases. Conclusion: HIV-2 indigenous cases had never been reported in China. These cases had brought new challenge on prevention, diagnosis and treatment of HIV/AIDS in China.

Evaluation of the vitros hiv combo 4th generation test for the identification of HIV infections.

BACKGROUND: Simultaneous detection of HIV 1 and 2 antibodies and HIV-1 p-24 antigen in the 4th generation tests is particularly effective for the identification of early acute HIV infections while maintaining accurate detection of long-established infections. OBJECTIVES: The aim of this study was to evaluate the new 4th generation VITROS HIV Combo test from Ortho-Clinical Diagnostics by comparing its results with those obtained using a 3rd generation HIV 1/2 antibody test (VITROS Anti HIV 1 + 2 from Ortho-Clinical Diagnostics) and a 4th generation test (LIAISON XL HIV Ab/Ag, DiaSorin) currently used in the Microbiology Unit of Legnano Hospital. STUDY DESIGN: One thousand and three samples of the normal daily routine (Group 1) were analyzed simultaneously with the three systems. The concordant and discordant sample results were further tested using Western blot and HIV-RNA assay (Roche). One hundred samples (Group 2) of known HIV positive subjects (63 of subtype B, 37 subtype non-B, and 51 with positive viraemia) and 50 samples (Group 3) with indeterminate Western blot were also examined using the three systems. From Group 3, 24 samples were collected from patients diagnosed with acute infection. RESULTS: The overall agreement between the three systems was 99.4% (99.5% in group 1, 100% in group 2 and 96.6% in group 3) with a coefficient Fleiss Kappa of 0.9814. Notably, the VITROS HIV Combo test was positive in all known HIV positive samples of group 2 without any statistically significant difference in the values of the sample/cut off ratios between the B and non-B subtypes and between the positive and negative viraemia samples in established infections. The VITROS HIV Combo test was also positive in all samples of patients with acute infection in group 3. CONCLUSIONS: The VITROS HIV Combo test has shown comparable performance to the other two assays in use of 3rd and 4th generation tests and is able to correctly identify both acute and established HIV infections independently of viraemia and HIV subtype.

Robotic selection for the rapid development of stable CHO cell lines for HIV vaccine production.

The production of envelope glycoproteins (Envs) for use as HIV vaccines is challenging. The yield of Envs expressed in stable Chinese Hamster Ovary (CHO) cell lines is typically 10-100 fold lower than other glycoproteins of pharmaceutical interest. Moreover, Envs produced in CHO cells are typically enriched for sialic acid containing glycans compared to virus associated Envs that possess mainly high-mannose carbohydrates. This difference alters the net charge and biophysical properties of Envs and impacts their antigenic structure. Here we employ a novel robotic cell line selection strategy to address the problems of low expression. Additionally, we employed a novel gene-edited CHO cell line (MGAT1- CHO) to address the problems of high sialic acid content, and poor antigenic structure. We demonstrate that stable cell lines expressing high levels of gp120, potentially suitable for biopharmaceutical production can be created using the MGAT1- CHO cell line. Finally, we describe a MGAT1- CHO cell line expressing A244-rgp120 that exhibits improved binding of three major families of bN-mAbs compared to Envs produced in normal CHO cells. The new strategy described has the potential to eliminate the bottleneck in HIV vaccine development that has limited the field for more than 25 years.