Distinct conformations of the HIV-1 V3 loop crown are targetable for broad neutralization

The V3 loop of the HIV-1 envelope (Env) protein elicits a vigorous, but largely non-neutralizing antibody response directed to the V3-crown, whereas rare broadly neutralizing antibodies (bnAbs) target the V3-base. Challenging this view, we present V3-crown directed broadly neutralizing Designed Ankyrin Repeat Proteins (bnDs) matching the breadth of V3-base bnAbs. While most bnAbs target prefusion Env, V3-crown bnDs bind open Env conformations triggered by CD4 engagement. BnDs achieve breadth by focusing on highly conserved residues that are accessible in two distinct V3 conformations, one of which resembles CCR5-bound V3. We further show that these V3-crown conformations can, in principle, be attacked by antibodies. Supporting this conclusion, analysis of antibody binding activity in the Swiss 4.5 K HIV-1 cohort (n = 4,281) revealed a co-evolution of V3-crown reactivities and neutralization breadth. Our results indicate a role of V3-crown responses and its conformational preferences in bnAb development to be considered in preventive and therapeutic approaches.

The number of samples from HIV infected individuals is predetermined through the size of the Swiss HIV-1 Cohort (SHCS) and the Zurich Primary HIV Infection Study (ZPHI) cohort as well as selection criteria applied for the Swiss 4.5K Screen (Rusert et al., Nat. Medecine, 2016).
No data was excluded.
In the luminex-based binding assay of IgG in human plasma to HIV-1 antigens ( Figure 6, Supplementary Figures 14 and 15), monoclonal HIV-1specific antibodies (PG09,2F5,19b,2G12,and HIV IG) were analyzed with each experiment as assay quality control (see also Kadelka et al., 2018). The accuracy of the test allowed that due to the large number of samples (4'281) and the limited availability of these historic clinical only single measurements without replicates were done as specified in (Kadelka et al., 2018). For all other experiments, all attempts at replication with optimized parameters as detailed in the materials and methods section were successful: DARPin binding ELISA ( Neutralization screens with a JR-CSF Env mutant panel: 128 JR-CSF mutant pseudoviruses carrying gp120 point mutations (Figure 4, Supplementary Tables 7 and 8) were probed for sensitivity to DARPins and selected mAbs in a two-step screening approach to detect resistance conferring mutants. In a first run, all mutant viruses of the JR-CSF Env panel were screened against all inhibitors. Mutants that passed in this first screen -a pre-set threshold of resistance (5-fold over IC50 against wt JR-CSF) against one DARPin -were followed up and retested against all DARPins, yielding in total at least two independent tests (Supplementary Table 8). Mutants that showed no effect against any of the inhibitors were not followed up and hence only tested once.
Mouse anti-FLAG® IgG1 antibody (Sigma Aldrich, clone M2, Cat#F1804 and Cat#F3165) was validated by the company for ELISA, Immunblotting, Immunprecipitation, Immunohistochemistry, Immuncytochemistry, and Immunfluorescence-Assays and has been optimized for detection of FLAG-tagged proteins in mammalian, plant and bacterial expression systems. The M2 antibody is not Calcium dependent (unlike clone M1) and is able to recognized the FLAG-tag at the N-terminus, C-terminus and at internal sites.
Alkaline phosphatase conjugated mouse anti-polyhistidin IgG2a antibody (Qiagen, clone HIS-1, Cat#A5588) was validated by the company for ELISA, Immunblotting and Dotblot-Assays. Anti-polyHistidine-Alkaline Phosphatase recognizes native as well as denatured / reduced forms of synthetic polyHistidine or polyHistidine-tagged fusion proteins. The product is reactive with fusion protein expressed by prokaryotic expression vectors and may be useful in various immunotechniques to identify the expression of a polyHistidine fusion protein in bacteria, bacterial lysates or cells and tissues transfected with a polyHistidine-tagged fusion protein  (2018)).
All HIV-1 Envelope protein specific antibodies were validated in the references provided in Supplementary Table 11 and their specificity was verified by the use of appropriate background controls in the current study.

HEK 293-T cells were obtained from the American Type Culture Collection and TZM-bl cells through the NIH AIDS Reagent
Program. HEK 293T FreestyleTM suspension (293F and Expi293F) cells were purchased from Thermo Fisher.
None of the cell lines used were authenticated again after reception from the specified original source.
In the Trkola laboratory, cell lines are routinely tested for mycoplasma contamination. No such contamination was detected in the cells used for the present study.
No commonly misidentified cell lines were used in the study.

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Outcomes
The SHCS, founded in 1988, is highly representative of the HIV epidemiology in Switzerland as it includes an estimated 53% of all HIV cases diagnosed in Switzerland since the onset of the epidemic, 72% of all patients receiving ART in Switzerland, and 69% of the nationwide registered AIDS cases. The Zurich Primary HIV Infection study (ZPHI) specifically enrolls patients with documented acute or recent primary HIV-1 infection. The following eight host, viral, and disease parameters were included in the analysis of the current study: viral load, CD4 count, infection length, virus diversity, gender, transmission mode, ethnicity, and viral pol subtype. These covariate-relevant population characteristics are provided as Supplementary Table 10 Table S2.
The Swiss HIV Cohort Study (SHCS) is a prospective, nationwide, longitudinal, non-interventional, observational, clinic-based cohort with semi-annual visits and blood collections, enrolling all HIV-infected adults living in Switzerland. The Zurich Primary HIV Infection study (ZPHI) is an ongoing, observational, non-randomized, single center cohort founded in 2002.
The SHCS and the ZPHI have been approved by the ethics committee of the participating institutions (Kantonale Ethikkommission Bern, Ethikkommission des Kantons St. Gallen, Comité départemental d'éthique des spécialités médicales et de médicine communautaire et de premier recours, Kantonale Ethikkommission Zürich, Repubblica e Cantone Ticino -Comitato Ethico Cantonale, Commission cantonale d'éthique de la recherche sur l'être humain, Ethikkommission beider Basel for the SHCS and Kantonale Ethikkommission Zürich for the ZPHI) and written informed consent had been obtained from all participants.
Detailed information on the SHCS and ZPHI cohorts is openly available on http://www.shcs.ch and http://www.ClinicalTrials.gov respectively.
The Swiss HIV Cohort Study (SHCS) is a prospective, nationwide, longitudinal, non-interventional, observational, clinic-based cohort with semi-annual visits and blood collections, enrolling all HIV-infected adults living in Switzerland. ZPHI study is a is an open label, non-randomized, observational, single center study at the University Hospital Zurich, Division of Infectious Diseases and Hospital Epidemiology. This study started in January 2002 and is estimated to be completed in January 2025. Both SHCS and ZPHI maintain a comprehensive, longitudinal, anonymous data collection of all participants, including extensive clinical and demographic data. The following data were used in the current study: longitudinal viral load and CD4 measurements, clinical history (antiretroviral drug history, infection length, and patient demographics), and pol nucleotide sequence data from genotypic antiretroviral drug resistance tests. The ethnicity (race) information is self-reported by the SHCS and ZPHI study participants at enrollment. Study nurses or study physicians use a structured interview questionnaire and participants are asked whether they belong to one of the following races/ethnicities: white, black, Hispano-American, or Asian. These categories cover the major ethnicities in Switzerland. Health care access is guaranteed for all races/ethnicities living in Switzerland and the same is true for participation in the SHCS and the ZPHI. Please see the supplementary note of Rusert et al. (2016) for further details on how patient data were recorded. The following eight host, viral, and disease parameters were included in the analysis of the current study: viral load, CD4 count, infection length, virus diversity, gender, transmission mode, ethnicity, and viral pol subtype. These covariate-relevant population characteristics are provided as Supplementary Table 10 Table S2.
The primary outcome measure of the ZPHI is to evaluate the effect of early-cART on the viral setpoint, which however is not relevant to the present study.
Here neutralization breadth was used as a categorical response variable. Patients were defined to have neutralization breadth if their plasma reached cross-, broad-or elite-neutralization activity score as determined in Rusert et al., Nat. Med., 2016 andKouyos et al., Nature, 2018. Patients with no or weak neutralization activity scores were categorized as having no neutralization. Viral load and CD4 level (both measured at time of sampling), viral pol diversity and infection length were included as continuous variables. The remaining four factors were used as categorical variables and analyzed in relation to the reference category (sex: reference male; mode of transmission: reference men having sex with men (MSM); ethnicity: reference White; HIV-1 clade: reference clade B, neutralization breadth: reference no neutralization).