Determinants and Dynamics of SARS-CoV-2 Infection in a Diverse Population: 6-Month Evaluation of a Prospective Cohort Study.

BACKGROUND: We studied risk factors, antibodies, and symptoms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in a diverse, ambulatory population. METHODS: A prospective cohort (n = 831) previously undiagnosed with SARS-CoV-2 infection underwent serial testing (SARS-CoV-2 polymerase chain reaction, immunoglobulin G [IgG]) for 6 months. RESULTS: Ninety-three participants (11.2%) tested SARS-CoV-2-positive: 14 (15.1%) asymptomatic, 24 (25.8%) severely symptomatic. Healthcare workers (n = 548) were more likely to become infected (14.2% vs 5.3%; adjusted odds ratio, 2.1; 95% confidence interval, 1.4-3.3) and severely symptomatic (29.5% vs 6.7%). IgG antibodies were detected after 79% of asymptomatic infections, 89% with mild-moderate symptoms, and 96% with severe symptoms. IgG trajectories after asymptomatic infections (slow increases) differed from symptomatic infections (early peaks within 2 months). Most participants (92%) had persistent IgG responses (median 171 days). In multivariable models, IgG titers were positively associated with symptom severity, certain comorbidities, and hospital work. Dyspnea and neurologic changes (including altered smell/taste) lasted ≥ 120 days in ≥ 10% of affected participants. Prolonged symptoms (frequently more severe) corresponded to higher antibody levels. CONCLUSIONS: In a prospective, ethnically diverse cohort, symptom severity correlated with the magnitude and trajectory of IgG production. Symptoms frequently persisted for many months after infection.Clinical Trials Registration. NCT04336215.

A Novel Sensitive Immunoassay Targeting the 5-Methylthio-d-Xylofuranose-Lipoarabinomannan Epitope Meets the WHO's Performance Target for Tuberculosis Diagnosis.

The only currently commercialized point-of-care assay for tuberculosis (TB) that measures lipoarabinomannan (LAM) in urine (Alere LF-LAM) has insufficient sensitivity. We evaluated the potential of 100 novel monoclonal antibody pairs targeting a variety of LAM epitopes on a sensitive electrochemiluminescence platform to improve the diagnostic accuracy. In the screening, many antibody pairs showed high reactivity to purified LAM but performed poorly at detecting urinary LAM in clinical samples, suggesting differences in antigen structure and immunoreactivity of the different LAM sources. The 12 best antibody pairs from the screening were tested in a retrospective case-control study with urine samples from 75 adults with presumptive TB. The best antibody pair reached femtomolar analytical sensitivity for LAM detection and an overall clinical sensitivity of 93% (confidence interval [CI], 80% to 97%) and specificity of 97% (CI, 85% to 100%). Importantly, in HIV-negative subjects positive for TB by sputum smear microscopy, the test achieved a sensitivity of 80% (CI, 55% to 93%). This compares to an overall sensitivity of 33% (CI, 20% to 48%) of the Alere LF-LAM and a sensitivity of 13% (CI, 4% to 38%) in HIV-negative subjects in the same sample set. The capture antibody targets a unique 5-methylthio-d-xylofuranose (MTX)-dependent epitope in LAM that is specific to the Mycobacterium tuberculosis complex and shows no cross-reactivity with fast-growing mycobacteria or other bacteria. The present study provides evidence that improved assay methods and reagents lead to increased diagnostic accuracy. The results of this work have informed the development of a sensitive and specific novel LAM point-of-care assay with the aim to meet the WHO's performance target for TB diagnosis.

Characterization of structural features and diversity of variable-region determinants of related quaternary epitopes recognized by human and rhesus macaque monoclonal antibodies possessing unusually potent neutralizing activities.

A series of potently neutralizing monoclonal antibodies (MAbs) that target quaternary epitopes on the native Env trimer have recently been described. A common feature shared by these antibodies is the critical involvement of sites in both the V2 and V3 variable domains in antibody recognition. In this study the gp120 variable-region determinants were mapped for eight rhesus macaque monoclonal antibodies (RhMAbs) possessing potently neutralizing activity specific for a quaternary target in SF162 Env and compared to those originally identified for human MAb 2909. These studies showed that determinants for the epitopes defined by the RhMAbs differed in both the V2 (positions 160, 167, and 169) and V3 (positions 313 and 315) regions from 2909, and in a number of cases, from each other. Attempts to reconstitute expression of these epitopes on the cell surface by cotransfecting Envs containing either the V2 or the V3 determinant of the epitope were not successful, suggesting that these epitopes were expressed on individual protomers in a trimer-dependent manner. Several of the V2 positions found to be critical for expression of these quaternary epitopes also significantly affected exposure and neutralization sensitivity of targets in the V3 and CD4-binding domains. These results demonstrated a considerable diversity in the fine structure of this class of epitopes and further suggested a potentially important relationship between the expression of such quaternary epitopes and V1/V2-mediated masking of immunodominant epitopes.

Potent and broad neutralization of HIV-1 subtype C by plasma antibodies targeting a quaternary epitope including residues in the V2 loop.

The targets of broadly cross-neutralizing (BCN) antibodies are of great interest in the HIV vaccine field. We have identified a subtype C HIV-1-superinfected individual, CAP256, with high-level BCN activity, and characterized the antibody specificity mediating breadth. CAP256 developed potent BCN activity peaking at 3 years postinfection, neutralizing 32 (76%) of 42 heterologous viruses, with titers of antibodies against some viruses exceeding 1:10,000. CAP256 showed a subtype bias, preferentially neutralizing subtype C and A viruses over subtype B viruses. CAP256 BCN serum targeted a quaternary epitope which included the V1V2 region. Further mapping identified residues F159, N160, L165, R166, D167, K169, and K171 (forming the FN/LRD-K-K motif) in the V2 region as crucial to the CAP256 epitope. However, the fine specificity of the BCN response varied over time and, while consistently dependent on R166 and K169, became gradually less dependent on D167 and K171, possibly contributing to the incremental increase in breadth over 4 years. The presence of an intact FN/LRD-K-K motif in heterologous viruses was associated with sensitivity, although the length of the adjacent V1 loop modulated the degree of sensitivity, with a shorter V1 region significantly associated with higher titers. Repair of the FN/LRD-K-K motif in resistant heterologous viruses conferred sensitivity, with titers sometimes exceeding 1:10,000. Comparison of the CAP256 epitope with that of the PG9/PG16 monoclonal antibodies suggested that these epitopes overlapped, adding to the mounting evidence that this may represent a common neutralization target that should be further investigated as a potential vaccine candidate.

Lipoarabinomannan antigenic epitope differences in tuberculosis disease subtypes.

An accurate urine test for diverse populations with active tuberculosis could be transformative for preventing TB deaths. Urinary liporabinomannan (LAM) testing has been previously restricted to HIV co-infected TB patients. In this study we evaluate urinary LAM in HIV negative, pediatric and adult, pulmonary and extrapulmonary tuberculosis patients. We measured 430 microbiologically confirmed pretreatment tuberculosis patients and controls from Peru, Guinea Bissau, Venezuela, Uganda and the United States using three monoclonal antibodies, MoAb1, CS35, and A194, which recognize distinct LAM epitopes, a one-sided immunoassay, and blinded cohorts. We evaluated sources of assay variability and comorbidities (HIV and diabetes). All antibodies successfully discriminated TB positive from TB negative patients. ROAUC from the average of three antibodies' responses was 0.90; 95% CI 0.87-0.93, 90% sensitivity, 73.5% specificity (80 pg/mL). MoAb1, recognizing the 5-methylthio-D-xylofuranose(MTX)-mannose(Man) cap epitope, performed the best, was less influenced by glycosuria and identified culture positive pediatric (N = 19) and extrapulmonary (N = 24) patients with high accuracy (ROAUC 0.87, 95% CI 0.77-0.98, 0.90 sensitivity 0.80 specificity at 80 pg/mL; ROAUC = 0.96, 95% CI 0.92-0.99, 96% sensitivity, 80% specificity at 82 pg/mL, respectively). The MoAb1 antibody, recognizing the MTX-Man cap epitope, is a novel analyte for active TB detection in pediatric and extrapulmonary disease.

Detection of lipoarabinomannan in urine and serum of HIV-positive and HIV-negative TB suspects using an improved capture-enzyme linked immuno absorbent assay and gas chromatography/mass spectrometry.

TB diagnosis and treatment monitoring in resource limited regions rely heavily on serial sputum smear microscopy and bacterial culture. These microbiological methods are time-consuming, expensive and lack adequate sensitivity. The WHO states that improved TB diagnosis and treatment is imperative to achieve an end to the TB epidemic by 2030. Commercially available lipoarabinomannan (LAM) detection tools perform at low sensitivity that are highly dependent on the underlying immunological status of the patient; those with advanced HIV infection perform well. In this study, we have applied two novel strategies towards the sensitive diagnosis of TB infection based on LAM: Capture ELISA to detect LAM in paired urine and serum samples using murine and human monoclonal antibodies, essentially relying on LAM as an 'immuno-marker'; and, secondly, detection of α-d-arabinofuranose and tuberculostearic acid (TBSA)- 'chemical-markers' unique to mycobacterial cell wall polysaccharides/lipoglycans by our recently developed gas chromatography/mass spectrometry (GC/MS) method. Blinded urine specimens, with microbiologically confirmed active pulmonary TB or non TB (HIV+/HIV-) were tested by the aforementioned assays. LAM in patient urine was detected in a concentration range of 3-28 ng/mL based on GC/MS detection of the two LAM-surrogates, d-arabinose and tuberculostearic acid (TBSA) correctly classifying TB status with sensitivity > 99% and specificity = 84%. The ELISA assay had high sensitivity (98%) and specificity (92%) and the results were in agreement with GC/MS analysis. Both tests performed well in their present form particularly for HIV-negative/TB-positive urine samples. Among the HIV+/TB+ samples, 52% were found to have >10 ng/mL urinary LAM. The detected amounts of LAM present in the urine samples also appears to be associated with the gradation of the sputum smear, linking elevated LAM levels with higher mycobacterial burden (odds ratio = 1.08-1.43; p = 0.002). In this small set, ELISA was also applied to parallel serum samples confirming that serum could be an additional reservoir for developing a LAM-based immunoassay for diagnosis of TB.

Antibodies that are cross-reactive for human immunodeficiency virus type 1 clade a and clade B v3 domains are common in patient sera from Cameroon, but their neutralization activity is usually restricted by epitope masking.

Sera from human immunodeficiency virus type 1 (HIV-1)-infected North American patients recognized a fusion protein expressing a V3 loop from a clade B primary isolate virus (JR-CSF) but not from a clade A primary isolate virus (92UG037.8), while most sera from Cameroonian patients recognized both fusion proteins. Competition studies of consensus V3 peptides demonstrated that the majority of the cross-reactive Cameroonian sera contained cross-reactive antibodies that reacted strongly with both V3 sequences. V3-specific antibodies purified from all six cross-reactive sera examined had potent neutralizing activity for virus pseudotyped with envelope proteins (Env) from SF162, a neutralization-sensitive clade B primary isolate. For four of these samples, neutralization of SF162 pseudotypes was blocked by both the clade A and clade B V3 fusion proteins, indicating that this activity was mediated by cross-reactive antibodies. In contrast, the V3-reactive antibodies from only one of these six sera had significant neutralizing activity against viruses pseudotyped with Envs from typically resistant clade B (JR-FL) or clade A (92UG037.8) primary isolates. However, the V3-reactive antibodies from these cross-reactive Cameroonian sera did neutralize virus pseudotyped with chimeric Envs containing the 92UG037.8 or JR-FL V3 sequence in Env backbones that did not express V1/V2 domain masking of V3 epitopes. These data indicated that Cameroonian sera frequently contain cross-clade reactive V3-directed antibodies and indicated that the typical inability of such antibodies to neutralize typical, resistant primary isolate Env pseudotypes was primarily due to indirect masking effects rather than to the absence of the target epitopes.

The C108g epitope in the V2 domain of gp120 functions as a potent neutralization target when introduced into envelope proteins derived from human immunodeficiency virus type 1 primary isolates.

Monoclonal antibodies (MAbs) directed against epitopes in the V2 domain of human immunodeficiency virus type 1 gp120 often possess neutralizing activity, but these generally are highly type specific, neutralize only laboratory isolates, or have low potency. The most potent of these is C108g, directed against a type-specific epitope in HXB2 and BaL gp120s, which is glycan dependent and, in contrast to previous reports, dependent on intact disulfide bonds. This epitope was introduced into two primary Envs, derived from a neutralization-sensitive (SF162) and a neutralization-resistant (JR-FL) isolate, by substitution of two residues and, for SF162, addition of an N-linked glycosylation site. C108g effectively neutralized both variant Envs with considerably higher potency than standard MAbs against the V3 and CD4-binding domains and the broadly neutralizing MAbs 2G12 and 2F5. These amino acid substitutions also introduced the epitope recognized by a second V2-specific MAb, 10/76b, but this MAb possessed potent neutralizing activity only in the absence of the glycan required for C108g reactivity. In contrast to other gp120-specific neutralizing MAbs, C108g did not block binding of soluble Env proteins to either the CD4 or the CCR5 receptor, but studies with a fusion-arrested Env indicated that C108g neutralized at a step preceding the one blocked by the gp41-specific MAb, 2F5. These results indicate that the V1/V2 domain possesses targets that mediate potent neutralization of primary viral isolates via a novel mechanism and suggest that inclusion of carbohydrate determinants into these epitopes may help overcome the indirect masking effects that limit the neutralizing potency of antibodies commonly produced after infection.

The V1/V2 domain of gp120 is a global regulator of the sensitivity of primary human immunodeficiency virus type 1 isolates to neutralization by antibodies commonly induced upon infection.

A major problem hampering the development of an effective vaccine against human immunodeficiency virus type 1 (HIV-1) is the resistance of many primary viral isolates to antibody-mediated neutralization. To identify factors responsible for this resistance, determinants of the large differences in neutralization sensitivities of HIV-1 pseudotyped with Env proteins derived from two prototypic clade B primary isolates were mapped. SF162 Env pseudotypes were neutralized very potently by a panel of sera from HIV-infected individuals, while JR-FL Env pseudotypes were neutralized by only a small fraction of these sera. This differential sensitivity to neutralization was also observed for a number of monoclonal antibodies (MAbs) directed against sites in the V2, V3, and CD4 binding domains, despite often similar binding affinities of these MAbs towards the two soluble rgp120s. The neutralization phenotypes were switched for chimeric Envs in which the V1/V2 domains of these two sequences were exchanged, indicating that the V1/V2 region regulated the overall neutralization sensitivity of these Envs. These results suggested that the inherent neutralization resistance of JR-FL, and presumably of related primary isolates, is to a great extent mediated by gp120 V1/V2 domain structure rather than by sequence variations at the target sites. Three MAbs (immunoglobulin G-b12, 2G12, and 2F5) previously reported to possess broad neutralizing activity for primary HIV-1 isolates neutralized JR-FL virus at least as well as SF162 virus and were not significantly affected by the V1/V2 domain exchanges. The rare antibodies capable of neutralizing a broad range of primary isolates thus appeared to be targeted to exceptional epitopes that are not sensitive to V1/V2 domain regulation of neutralization sensitivity.

Efficient isolation of novel human monoclonal antibodies with neutralizing activity against HIV-1 from transgenic mice expressing human Ig loci.

Despite considerable interest in the isolation of mAbs with potent neutralization activity against primary HIV-1 isolates, both for identifying useful targets for vaccine development and for the development of therapeutically useful reagents against HIV-1 infection, a relatively limited number of such reagents have been isolated to date. Human mAbs (hu-mAbs) are preferable to rodent mAbs for treatment of humans, but isolation of hu-mAbs from HIV-infected subjects by standard methods of EBV transformation of B cells or phage display of Ig libraries is inefficient and limited by the inability to control or define the original immunogen. An alternative approach for the isolation of hu-mAbs has been provided by the development of transgenic mice that produce fully hu-mAbs. In this report, we show that immunizing the XenoMouse G2 strain with native recombinant gp120 derived from HIV(SF162) resulted in robust humoral Ab responses against gp120 and allowed the efficient isolation of hybridomas producing specific hu-mAbs directed against multiple regions and epitopes of gp120. hu-mAbs possessing strong neutralizing activity against the autologous HIV(SF162) strain were obtained. The epitopes recognized were located in three previously described neutralization domains, the V2-, V3- and CD4-binding domains, and in a novel neutralization domain, the highly variable C-terminal region of the V1 loop. This is the first report of neutralizing mAbs directed at targets in the V1 region. Furthermore, the V2 and V3 epitopes recognized by neutralizing hu-mAbs were distinct from those of previously described human and rodent mAbs and included an epitope requiring a full length V3 loop peptide for effective presentation. These results further our understanding of neutralization targets for primary, R5 HIV-1 viruses and demonstrate the utility of the XenoMouse system for identifying new and interesting epitopes on HIV-1.

Human monoclonal antibodies specific for conformation-sensitive epitopes of V3 neutralize human immunodeficiency virus type 1 primary isolates from various clades.

The epitopes of the V3 domain of the human immunodeficiency virus type 1 (HIV-1) gp120 glycoprotein have complex structures consisting of linear and conformational antigenic determinants. Anti-V3 antibodies (Abs) recognize both types of elements, but Abs which preferentially react to the conformational aspect of the epitopes may have more potent neutralizing activity against HIV-1, as recently suggested. To test this hypothesis, human anti-V3 monoclonal Abs (MAbs) were selected using a V3 fusion protein (V3-FP) which retains the conformation of the third variable region. The V3-FP consists of the V3(JR-CSF) sequence inserted into a truncated form of murine leukemia virus gp70. Six human MAbs which recognize epitopes at the crown of the V3 loop were selected with the V3-FP. They were found to react more strongly with molecules displaying conformationally intact V3 than with linear V3 peptides. In a virus capture assay, these MAbs showed cross-clade binding to native, intact virions of clades A, B, C, D, and F. No binding was found to isolates from subtype E. The neutralizing activity of MAbs against primary isolates was determined in three assays: the GHOST cell assay, a phytohemagglutinin-stimulated peripheral blood mononuclear cell assay, and a luciferase assay. While these new MAbs displayed various degrees of activity, the pattern of cross-clade neutralization of clades A, B, and F was most pronounced. The neutralization of clades C and D viruses was weak and sporadic, and neutralization of clade E by these MAbs was not detected. Analysis by linear regression showed a highly significant correlation (P < 0.0001) between the strength of binding of these anti-V3 MAbs to intact virions and the percent neutralization. These studies demonstrate that human MAbs to conformation-sensitive epitopes of V3 display cross-clade reactivity in both binding to native, intact virions and neutralization of primary isolates.