Improving comparability of studies using HCoV-OC43 as a surrogate for SARS-CoV-2

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has renewed interest in human coronaviruses that cause the common cold, particularly as research with them at biosafety level (BSL)-2 avoids the added costs and biosafety concerns that accompany work with SARS-COV-2, BSL-3 research. One of these, human coronavirus OC43 (HCoV-OC43), is a well-matched surrogate for SARS-CoV-2 because it is also a Betacoronavirus, targets the human respiratory system, is transmitted via respiratory aerosols and droplets and is relatively resistant to disinfectants. Unfortunately, growth of HCoV-OC43 in the recommended human colon cancer (HRT-18) cells does not produce obvious cytopathic effect (CPE) and its titration in these cells requires expensive antibody-based detection. Consequently, multiple quantification approaches for HCoV-OC43 using alternative cell lines exist, which complicates comparison of research results. Hence, we investigated the basic growth parameters of HCoV-OC43 infection in three of these cell lines (HRT-18, human lung fibroblasts (MRC-5) and African green monkey kidney (Vero E6) cells) including the differential development of cytopathic effect (CPE) and explored reducing the cost, time and complexity of antibody-based detection assay. Multi-step growth curves were conducted in each cell type in triplicate at a multiplicity of infection of 0.1 with daily sampling for seven days. Samples were quantified by tissue culture infectious dose50(TCID50)/ml or plaque assay (cell line dependent) and additionally analyzed on the Sartorius Virus Counter 3100 (VC), which uses flow virometry to count the total number of intact virus particles in a sample. We improved the reproducibility of a previously described antibody-based detection based TCID50 assay by identifying commercial sources for antibodies, decreasing antibody concentrations and simplifying the detection process. The growth curves demonstrated that HCoV-O43 grown in MRC-5 cells reached a peak titer of ~107 plaque forming units/ml at two days post infection (dpi). In contrast, HCoV-OC43 grown on HRT-18 cells required six days to reach a peak titer of ~106.5 TCID50/ml. HCoV-OC43 produced CPE in Vero E6 cells but these growth curve samples failed to produce CPE in a plaque assay after four days. Analysis of the VC data in combination with plaque and TCID50 assays together revealed that the defective:infectious virion ratio of MRC-5 propagated HCoV-OC43 was less than 3:1 for 1-6 dpi while HCoV-OC43 propagated in HRT-18 cells varied from 41:1 at 1 dpi, to 329:4 at 4 dpi to 94:1 at 7 dpi. These results should enable better comparison of extant HCoV-OC43 study results and prompt further standardization efforts.

Dynamics of neutralizing antibody responses to SARS-CoV-2 in patients with COVID-19: an observational study

Our understanding of the protective immunity, particularly the long-term dynamics of neutralizing antibody (NAbs) response to SARS-CoV-2, is currently limited. We enrolled a cohort of 545 COVID-19 patients from Hubei, China, who were followed up up to 7 months, and determined the dynamics of NAbs to SARS-CoV-2 by using a surrogate virus neutralization test (sVNT). In our validation study, sVNT IC50 titers and the neutralization rate measured at a single dilution (1:20) were well correlated with FRNT titers (r = 0.85 and 0.84, respectively). The median time to seroconversion of NAbs was 5.5 days post onset of symptoms. The rate of positive sVNT was 52% in the first week, reached 100% in the third week, and remained above 97% till 6 months post onset. Quantitatively, NAbs peaked in the fourth week and only a quarter of patients had an estimated peak titer of >1000. NAbs declined with a half-time of 61 days (95% CI: 49–80 days) within the first two months, and the decay deaccelerated to a half-time of 104 days (95% CI: 86–130 days) afterward. The peak levels of NAbs were positively associated with severity of COVID-19 and age, while negatively associated with serum albumin levels. The observation that the low-moderate peak neutralizing activity and fast decay of NAbs in most naturally infected individuals called for caution in evaluating the feasibility of antibody-based therapy and vaccine durability. NAbs response positively correlated with disease severity, warning for the possibility of repeat infection in patients with mild COVID-19.

926. Antibody Response to HPV Vaccination in Pediatric and Adolescent People Living with HIV (PLWH)

Background Immune dysfunction related to HIV infection is associated with an inability to clear HPV infection and may compromise the immunogenicity of quadrivalent HPV vaccine Gardasil® (4v HPV). Methods Between 2005 and 2017, males and females 7 to 20 years old age, were offered 3-dose 4v HPV vaccine. Plasma IgG titers to HPV 6 (H6), 11 (H11), 16 (H16) and 18 (H18) were measured using multiplex VLP-based ELISA. For the 36 patients, median interval from 1st dose to 2nd and 3rd doses were 73 and 216 days. Plasma sample 1 was collected at median of 91 days after dose 1, sample 2, 169 and sample 3, 740 after respective vaccine doses. A 4th sample was available for 26 patients, median 2327 days after dose 1. Rank-sum test, Χ 2 or Fisher’s Exact Test were employed. Results Before vaccination, 10 (28%) were seropositive to 1 or more HPV types. The baseline seropositives were older than seronegatives (16 years vs 11; p=0.007). After dose 3 all participants had an Ab response to at least 1 HPV type and 32 (89%) were seropositive for 4 HPV types. Seroconversions were H18, 87%; H16 97%; H11, 100%; H6, 97%. Seroconversions after 1 dose of 4v HPV among the baseline seronegatives were 61%, 90%, 86% and 86%, respectively and 22 became seropositive for all 4 types. The 4 baseline seronegative PLWH with partial seroconversion had higher median HIV viral load (VL) compared to baseline seronegative group with full seroconversion (12,920 vs 101 copies/ml; p = 0.052), but had comparable CD4 counts. The rate of post vaccination seropositivity and baseline to peak titer response for each HPV type was not significantly different for baseline sero-groups. Among baseline seronegative, all 19 sampled distant from vaccination remained seropositive to at least 1 HPV type (84% to 3 or more types) and 6 (32%) became seronegative (sero-reversion). Those showing sero-reversion had higher VL compared to the 14 who remained seropositive (9100 vs 48; p =0.015). Time from last dose of 4v HPV to sample 4, CD4%, age, gender, and race/ethnicity were similar between the groups. Bar Graphs representing Ab response to the 4 HPV types following each dose of 4v HPV vaccine Conclusion In the complex environment of a pediatric HIV specialty clinic, most PLWH mounted Ab responses to 4v HPV that were durable. H18 was least immunogenic. Patients with higher HIV VL were less likely to seroconvert for all types and were more likely to sero-revert. Disclosures All Authors: No reported disclosures

Interim Analysis of the Extension Study with rVIII-SingleChain in Previously Untreated Patients (PUPs) with Severe Hemophilia A (CSL627-3001)

Background: rVIII-SingleChain is a novel B-domain truncated recombinant Factor VIII comprised of covalently bonded FVIII heavy and light chains designed to have a high binding affinity to von Willebrand factor. Aims: This multicenter, open-label, phase III extension study investigates the safety and efficacy of rVIII-SingleChain for prophylaxis and on-demand treatment of bleeding episodes in 50 previously untreated patients (PUPs) for at least 50 Exposure Days (EDs). An ITI substudy was implemented to allow the use of rVIII-SingleChain to attempt inhibitor eradication for PUPs who develop an inhibitor to FVIII. Method: PUPs with severe hemophilia A (no prior exposure to any FVIII product, and endogenous Factor VIII <1%) were assigned by the investigator to a prophylaxis or on-demand treatment regimen. Inhibitors were assessed monthly. Patients diagnosed with an inhibitor to FVIII (two consecutive central laboratory [CL] results of ≥0.6 BU/mL) could be enrolled into the ITI substudy or remain in the main study. The ITI substudy regimens are: 50 IU/kg 3x/week (low), 100 IU/kg daily (medium), or 200 IU/kg daily (high). Inhibitor eradication was defined as two consecutive CL results of <0.6 BU/mL. One subject was withdrawn per protocol due to high titer inhibitor diagnosis prior to ITI substudy implementation. Results: As of March 28, 2019, 23 PUPs were treated with rVIII-SingleChain. Median age: 1 y (range 0-5). Mean (SD) time on study: 21.6 (12.6) months. Race distribution; Asian 2, Black 7, White 14. There have been 147 bleeding events treated with rVIII-SingleChain rated for hemostatic efficacy by the investigator. While patients were inhibitor negative, the overall treatment success (rating of excellent/good) was 93%, and the annualized spontaneous bleeding rate (AsBR) was 0.58. The adverse event profile was as expected, as the most frequently occurring adverse events were upper respiratory tract infections and inhibitors. Twelve subjects (52%) [95% CI 31%, 73%] were diagnosed with an inhibitor to FVIII; 6 (26%) high titer (peak titer ≥5 BU/mL), and 6 (26%) low titer (peak titer <5 BU/mL). Seven of 11 inhibitor negative subjects achieved >50 EDs, 1 achieved 47 EDs, and 3 achieved <20 EDs. The median ED for inhibitor development (initial result) was 10, range 4-23. All PUPs enrolled had ≥1 risk factor for inhibitor development (Table 1) including genetic mutation, age of first exposure, initial treatment reason and assigned regimen, as well as bleeding events and infections; inhibitor positive and negative subjects were comparable. Of the 12 inhibitor positive subjects, 11 continued treatment with rVIII-SingleChain, 7 were treated with approximately 50 IU/kg 3x/week (low dose ITI), 3 with an increased prophylaxis regimen, 1 with no change in regimen, and 0 with high or medium dose ITI regimen (Table 2). Eight of 11 (73%) inhibitor positive subjects (2 high titer, 6 low titer) achieved eradication; 5 low titer subjects were eradicated within 6 months. The clinically relevant inhibitor subjects (2 high titer, and 1 persistent low titer) achieved eradication in a median of 15.7 months, 2 using low dose ITI, and 1 using increased prophylaxis. Eradicated patients were negative for a median of 13.6 months, and no inhibitor relapse was observed. Three remaining inhibitor positive patients are early in their rVIII-SingleChain inhibitor treatment (2.1, 3.5, and 5.4 months). Detailed analysis of the antibody signature was performed, and revealed epitope isotypes and subclass distribution comparable to other FVIII molecules. Conclusion: Overall, rVIII-SingleChain demonstrates a positive benefit:risk profile for safety and efficacy in PUPs. The crude high titer inhibitor rate is 26% which is consistent with other rFVIII products, whereas the crude low titer inhibitor rate is currently 26% which is on the higher end in comparison to other rFVIII products. Immunological analyses suggest a low affinity antibody population in subjects with low titer inhibitors. The majority of subjects (73%) who continued treatment with rVIII-SingleChain achieved eradication on a low dose ITI or prophylaxis regimen of approximately 50 IU/kg 3x/week or less. Additional time on study for the currently enrolled subjects is required to determine the final inhibitor and eradication rates in PUPs treated with rVIII-SingleChain. Disclosures Santagostino: Pfizer: Consultancy, Speakers Bureau; Bayer: Consultancy, Speakers Bureau; Shire / Takeda: Consultancy, Speakers Bureau. Koenigs:Roche: Consultancy; CSL Behring: Research Funding, Speakers Bureau; Bayer Vital GmbH: Research Funding, Speakers Bureau; Biotest AG: Research Funding, Speakers Bureau; Intersero: Research Funding; Grifols: Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Shire: Research Funding, Speakers Bureau; Sobi: Research Funding, Speakers Bureau. Djambas Khayat:Novo Nordisk: Consultancy, Speakers Bureau; Pfizer: Speakers Bureau. Lucas:CSL Behring: Employment. Salazar:CSL Behring: Employment. Brainsky:CSL Behring: Employment. Chung:CSL Behring: Employment. Goldstein:CSL Behring: Employment. Mahlangu:Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Spark: Consultancy, Honoraria, Research Funding, Speakers Bureau; Biomarin: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Sanofi: Research Funding; Unique: Research Funding; Catalyst Biosciences: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novo Nordisk: Consultancy, Honoraria, Research Funding, Speakers Bureau; Baxalta: Consultancy, Honoraria, Research Funding, Speakers Bureau; CSL Behring: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Mutations in Coronavirus Nonstructural Protein 10 Decrease Virus Replication Fidelity

ABSTRACTCoronaviruses (CoVs) are unique in encoding a 3′→5′ exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is required for high-fidelity replication, likely via proofreading. nsp14 associates with the CoV RNA-dependent RNA polymerase (nsp12-RdRp), and nsp14-ExoN activity is enhanced by binding nsp10, a small nonenzymatic protein. However, it is not known whether nsp10 functions in the regulation of CoV replication fidelity. To test this, we engineered single and double alanine substitution mutations into the genome of murine hepatitis virus (MHV-A59) containing ExoN activity [ExoN(+)] at positions within nsp10 known to disrupt the nsp10-nsp14 interactionin vitro. We show that an nsp10 mutant, R80A/E82A-ExoN(+), was five to ten times more sensitive to treatment with the RNA mutagen 5-fluorouracil (5-FU) than wild-type (WT)-ExoN(+), suggestive of decreased replication fidelity. This decreased-fidelity phenotype was confirmed using two additional nucleoside analogs, 5-azacytidine and ribavirin. R80A/E82A-ExoN(+) reached a peak titer similar to and demonstrated RNA synthesis kinetics comparable to those seen with WT-ExoN(+). No change in 5-FU sensitivity was observed for R80A/E82A-ExoN(−) relative to MHV-ExoN(−), indicating that the decreased-fidelity phenotype of R80A/E82A-ExoN(−) is linked to the presence of ExoN activity. Our results demonstrate that nsp10 is important for CoV replication fidelity and support the hypothesis that nsp10 functions to regulate nsp14-ExoN activity during virus replication.IMPORTANCEThe adaptive capacity of CoVs, as well as all other RNA viruses, is partially attributed to the presence of extensive population genetic diversity. However, decreased fidelity is detrimental to CoV replication and virulence; mutant CoVs with decreased replication fidelity are attenuated and more sensitive to inhibition by RNA mutagens. Thus, identifying the viral protein determinants of CoV fidelity is important for understanding CoV replication, pathogenesis, and virulence. In this report, we show that nsp10, a small, nonenzymatic viral protein, contributes to CoV replication fidelity. Our data support the hypothesis that CoVs have evolved multiple proteins, in addition to nsp14-ExoN, that are responsible for maintaining the integrity of the largest known RNA genomes.

Limited replication of yellow fever 17DD and 17D-Dengue recombinant viruses in rhesus monkeys

For the development of safe live attenuated flavivirus vaccines one of the main properties to be established is viral replication. We have used real-time reverse transcriptase-polymerase chain reaction and virus titration by plaque assay to determine the replication of yellow fever 17DD virus (YFV 17DD) and recombinant yellow fever 17D viruses expressing envelope proteins of dengue virus serotypes 2 and 4 (17D-DENV-2 and 17D-DENV-4). Serum samples from rhesus monkeys inoculated with YFV 17DD and 17D-DENV chimeras by intracerebral or subcutaneous route were used to determine and compare the viremia induced by these viruses. Viral load quantification in samples from monkeys inoculated by either route with YFV 17DD virus suggested a restricted capability of the virus to replicate reaching not more than 2.0 log10 PFU mL-1 or 3.29 log10 copies mL-1. Recombinant 17D-dengue viruses were shown by plaquing and real-time PCR to be as attenuated as YF 17DD virus with the highest mean peak titer of 1.97 log10 PFU mL-1 or 3.53 log10 copies mL-1. These data serve as a comparative basis for the characterization of other 17D-based live attenuated candidate vaccines against other diseases.

Immune Tolerance Induction in 28 Children with Hemophilia Awith Inhibitors.

Aim: To study the characteristics, treatment, and outcome of patients with hemophilia with inhibitors who have undergone immune tolerance induction (ITI) at the Children’s Hospital of Michigan over the past 14 years. Methods: In compliance with local IRB regulations, patient charts and laboratory databases were reviewed and salient data extracted. 28 boys underwent 29 attempts at immune tolerance induction. Results: Hemophilia A 26 boys with severe hemophilia A with inhibitors underwent 27 trials of ITI. In this cohort of 26 patients the average age at which patients developed an inhibitor was 22 months and the average age at start of ITI was 5 years 4 months (range 2 months to 17 years 5 months). The average number of exposure days prior to inhibitor development was 10 (1–47). The average time between development of an inhibitor and initiation of ITI was 43 months, with no difference between those who successfully completed ITI and those who did not. Six patients had low titer inhibitors (0.8–6.5 BU) and successfully completed ITI using a modified low dose ITI regimen of factor infusions 3–7 times per week. 20 of the patients with high titer inhibitors (6.4–1280 BU) were treated with daily infusions of 50–200 units/kg/d of factor VIII (FVIII) products. For ITI, 4 patients received high purity plasma derived FVIII (PD-FVIII) and 21 received recombinant FVIII (rFVIII) and one received both. In patients who became tolerized to FVIII, the average time to achieve an inhibitor titer of 0 Bethesda Units (BU) was 211 days. In those who were unable to achieve tolerance, the average length of the trial was 263 days. 21 of the ITI trials employed a central venous catheter and in 5 patients ITI was stopped after removal of the line because of recurrent infections. 14 boys received FEIBA, rFVIIa, or porcine FVIII for bleeding episodes during ITI; 8 of them failed ITI and one is still on therapy. Seven trials of ITI were in Caucasian patients (26 %), 17 in African American (AA) (63 %), and 3 in Middle Eastern patients (11 %). 19 patients achieved complete tolerance (73 %), 6 patients failed (23 %), one failed twice, and one patient continues on therapy. All but 2 patients who successfully completed ITI went on prophylaxis with FVIII. All patients who successfully completed ITI have maintained tolerance with a mean follow-up of 101 months (range 7–168). Table I: Hemophilia A Failed ITI Successful ITI *One still ongoing Number of trials* 7 (23 %) 19 (73 %) African Americans 7 (41 %) 10 (59 %) Non-AA 0 10 (100 %) Historical Peak Titer (mean) 345 BU 47 BU Titer at Start of ITI (mean) 62 BU 5 BU Peak Titer on ITI (mean) 168 BU 46 BU Age at inhibitor development (mean) 26 months 12 months ITI with PD-FVIII 1 4 ITI with rFVIII 7 15 Hemophilia B During this time period, 2 boys with severe hemophilia B underwent ITI. Both had severe allergic reactions at the time of inhibitor development; both underwent desensitization successfully but both failed ITI. Both started ITI with plasma derived factor nine at age 15 months. One developed nephrotic syndrome while on ITI. Conclusions: Most patients with Hemophilia A were able to achieve and maintain tolerance (73%). Higher titer inhibitors, hemophilia B, younger age at development of inhibitor, AA race and treatment of bleeds with bypass agents or porcine factor while on ITI were risk factors for ITI failures. Loss of central venous access with recurrent infections was also a common reason for ITI failure.

Chimeric Recombinant Human Metapneumoviruses with the Nucleoprotein or Phosphoprotein Open Reading Frame Replaced by That of Avian Metapneumovirus Exhibit Improved Growth In Vitro and Attenuation In Vivo

ABSTRACT Chimeric versions of recombinant human metapneumovirus (HMPV) were generated by replacing the nucleoprotein (N) or phosphoprotein (P) open reading frame with its counterpart from the closely related avian metapneumovirus (AMPV) subgroup C. In Vero cells, AMPV replicated to an approximately 100-fold-higher titer than HMPV. Surprisingly, the N and P chimeric viruses replicated to a peak titer that was 11- and 25-fold higher, respectively, than that of parental HMPV. The basis for this effect is not known but was not due to obvious changes in the efficiency of gene expression. AMPV and the N and P chimeras were evaluated for replication, immunogenicity, and protective efficacy in hamsters. AMPV was attenuated compared to HMPV in this mammalian host on day 5 postinfection, but not on day 3, and only in the nasal turbinates. In contrast, the N and P chimeras were reduced approximately 100-fold in both the upper and lower respiratory tract on day 3 postinfection, although there was little difference by day 5. The N and P chimeras induced a high level of neutralizing serum antibodies and protective efficacy against HMPV; AMPV was only weakly immunogenic and protective against HMPV challenge, reflecting antigenic differences. In African green monkeys immunized intranasally and intratracheally, the mean peak titer of the P chimera was reduced 100- and 1,000-fold in the upper and lower respiratory tracts, whereas the N chimera was reduced only 10-fold in the lower respiratory tract. Both chimeras were comparable to wild-type HMPV in immunogenicity and protective efficacy. Thus, the P chimera is a promising live HMPV vaccine candidate that paradoxically combines improved growth in vitro with attenuation in vivo.

Serum Bactericidal Test as a Prognostic Indicator in Acute Pulmonary Exacerbations of Cystic Fibrosis

The serum bactericidal test has been used for many years for optimal assessment of the efficacy of antibiotic therapy in patients with infective endocarditis and other bacterial infections. Its capacity to predict the bacteriological outcome of acute pulmonary exacerbations in patients with cystic fibrosis was evaluated. A total of 54 courses of intravenous antibiotic therapy were analyzed in 22 patients, whose ages ranged from 4 months to 24 years (mean age: 10 years). The serum bactericidal activity of blood samples, taken at expected peak and trough antibiotic levels on day 4 of therapy, were determined against the potentially pathogenic strains isolated in sputum at the time of admission. For 104 isolates (64 Pseudomonas aeruginosa, 28 Staphulococcus aureus, and 12 Haemophilus influenzae strains), the peak and trough bactericidal titers were compared to bacteriological outcome. Bacteriological success was defined as a decrease of 2 log10 units or more in the bacterial density in sputum between days 0 and 7 of therapy. At peak antibiotic levels, serum bactericidal titers of 1:128 or more were 96% (all isolates) and 89% (P aeruginosa isolates), predictive of cure, whereas serum bactericidal titers of less than 1:16 were 100% predictive of failure for all infecting bacteria. In patients aged less than 18 years, the best peak titer for predicting success was 1:64, with a predictive value of 96% for titers of 1:64 or greater The peak titer that best predicted success in patients aged 18 years or more was 1:128, with a predictive value of only 83% for titers of 1:128 or greater. No trough serum bactericidal titer achieved statistical significance as a predictor of bacteriological outcome in the two patient age groups. Peak serum bactericidal titers of 1:64 or more (patients aged less than 18 years) and of 1:128 or more (patients aged 18 years or more) are recommended to provide optimal treatment of acute pulmonary exacerbations of cystic fibrosis.