Study On Detection Assay,Molecular And Serological Characteristics Of SARS-CoV-2 And HIV-1 Infection

BackgroundEmerging and re-emerging infectious diseases are of critical public health concern around the world,including the coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),acquired immunodeficiency syndrome(AIDS)by human immunodeficiency virus-1(HIV-1),and various virus infection.High-throughput SARS-CoV-2 serological assays are important for screening population infection and evaluating population immunity,and tracing the source and animal hosts of infection.Further characterization of the dynamics of viral load and specific antibodies against SARS-CoV-2 in COVID19 patients and evaluation of their clinical value are important for diagnosis,antiviral treatment,epidemiological investigation and vaccine development for COVID-19.Moreover,SARS-CoV-2 has evolved to have variants with different phenotypes,and caused multiple waves of COVID-19 pandemic,which highlight the importance of continuous surveillance of SARS-CoV-2 mutant viruses.Genome sequencing is still the gold standard technology for identifying SARS-CoV-2 variants,but may not be appropriate for routine screening especially in resource-limited settings.The quantitative reverse transcription-polymerase chain reaction(RT-qPCR)requires equipment,conditions,and trained personnel.A comprehensive RT-qPCR-based assay for the detection of SARS-CoV-2 variants has not yet been developed.Therefore,there is an urgent need to develop a simple,rapid,and cost-effective assays with excellent detection performance for screening and distinguishing major SARS-CoV-2 variants.Furthermore,HIV-1 is characterized by extensive genetic heterogeneity and rapid evolution,therefore,HIV-1 genetic diversity is increasing in China,but its association with disease progression remains to be elucidated.Although early initiation of antiretroviral therapy(ART)can slow disease progression and reduce HIV-1-specific antibody response,the characteristics of anti-HIV antibody response in patients with delayed ART have not yet been fully investigated.Therefore,the purpose of this study is to establish serological and molecular detection methods for SARS-CoV-2 and HIV-1 infection,and explore their serological and molecular characteristics and the relationship with disease prognosis.Methods1.A luciferase immunosorbent assay(LISA)for detection of IgG antibody against S ARS-CoV-2 nucleoprotein was developed.The diagnostic performance was evaluated in 492 and 88 serum sample collected from COVID-19 patients and healthy blood donors,respectively.2.Demographic and clinical information were obtained from 24 serve/critical and 76 mild/moderate COVID-19 patients.Serial samples of blood,nasal and pharyngeal and anal swabs were collected at different time points post-onset.SARS-CoV-2 RNA were measured by RT-qPCR,and anti-SARS-CoV-2 antibodies were detected by LISA and enzyme linked immunosorbent assay(ELISA),respectively.3.To designed and analytically validated the next-generation molecular detection and diagnosis technological platform based on CRISPR-Cas12a system constructed by clustered regularly interspaced short palindromic repeats(CRISPR)and Cas12a protein for detection of SARS-CoV-2 variants of concern(VOCs).The current study further evaluated the combination of reverse transcription-polymerase chain reaction(RT-PCR)and CRISPR-Cas12a to improve the detection sensitivity,and developed a universal system by introducing a protospacer adjacent motif(PAM)near the target mutation sites through PCR primer design to detect mutations without PAM.In regard to the new features of Omicron variant,the study design allele-specific CRISPR RNAs(crRNAs)targeting the signature mutations in the spike protein of Omicron variant,and to develop a CRISPR-Cas12a-based assay to specifically detect Omicron variant.SARSCoV-2 positive oropharyngeal swab specimens were used to evaluate the performance of the CRISPR-Cas12a-mediated assay to identify major SARS-CoV-2 variants of concern.4.Collected data in an observational longitudinal cohort of 860 HIV-1-infected Men who have sex with men(MSM)in Guangzhou,China between January 2008 and March 2017.Kaplan-Meier analysis and Cox proportional hazard model were used to predict the incidence or time from HIV-1 diagnosis to immunodeficiency progression(CD4 cell count<200 cells/μL)as well as adjusted hazard ratio(aHR).5.Demographic characteristics were collected from 81 chronic HIV-1-infected patients under ART and the longitudinally dynamic changes of HIV-1 RNA and DNA,CD4 and CD 8 counts,as well as 5 anti-HIV antibodies were analyzed.The factors associated with antibody decline were evaluated by binary logistic regression analysis.Results1.The fluorescence value detected by LISA in 88 healthy blood donor serum samples was no different from that of the blank control,and the specificity was 100%.In addition,the detection rate of anti-SARS-CoV-2 nucleoprotein IgG antibody was 42.7%(35/82)during 0-7 days post onset(d.p.o.),then increased to 71.2%(104/146)8-14 d.p.o.,96.7%(117/121)15-21 d.p.o.,98.4%(61/62)21-28 d.p.o.and 100.0%(81/81)more than 28 d.p.o..Furthermore,A dose-dependent reactivity was observed between antibodies against SARS-CoV nucleoprotein and SARS-CoV-2 nucleoprotein,but not the MERS-CoV nucleoprotein specific antibody.2.Respiratory SARS-CoV-2 RNA was detectable in 58.0%(58/100)COVID-19 patients after admission and lasted for a median of 15 days post-onset.In addition,5.9%(1/17)and 20.2%(19/94)of the blood and anal swab specimens were positive for SARS-CoV-2 RNA,respectively.Anal viral RNA was more frequently detected in the patients who were positive for viral RNA in the respiratory samples after admission.Specific anti-SARS-CoV-2 antibody developed within two weeks after onset,reached peak approximately 17 days post-onset and then maintained at relatively high level up to 50 days in most patients.However,the levels of antibodies were variable among the patients.High titers of antibodies are associated with the severity of the disease,the proportion of severe/critical COVID-19 patients is higher in the high antibody titer group than that in the low antibody titer group(36.2%vs 7.5%,P=0.002).3.The study results indicated that the CRISPR-Cas12a assay using corresponding specific crRNA could accurately detect the signature mutations(K417N/T,L452R/Q,T478K,E484K/Q,N501Y,D614G)in the spike protein of SARS-CoV-2,and the low limit of detection could reach 10 copies/reaction.Benchmarked against Sanger sequencing,the 8 allele-specific crRNAs analyzed can specifically identify the corresponding mutations with a positive predictive value of 83.3%-100%and a negative predictive value of 81.3%-100%.Additionally,as per the combined analysis of data detected using several designed crRNAs,the CRISPR-Cas12a-mediated assay distinguished wild-type and major variants of concern of SARS-CoV-2 with a sensitivity of 93.8%-100.0%and specificity of 100.0%.The detection results between Sanger sequencing and CRISPR-Cas12a assay showed a concordance of 98.1%(53/54).Moreover,there was no cross-reaction observed when detecting 33 SARS-CoV-2 negative clinical samples infected with common respiratory pathogens.4.CRISPR-Cas12a-based detection could accurately detect SARS-CoV-2 Omicron variant in 5 laboratory-confirmed clinical samples and distinguish them from 57 SARS-CoV-2 positive clinical samples infected with wild-type(N=8)and the variants of Alpha(N=17),Beta(N=17)and Delta(N=15).In addition,no cross-reaction was observed when detecting 16 clinical samples infected with 9 common respiratory pathogens.5.CRF01＿AE and HIV-1 subtype B infection were associated with larger proportion and higher incidence of immunodeficiency than infection with CRF07＿BC or CRF55＿01B.Compared with CRF07＿BC,the time and risk from HIV-1 diagnosis to immunodeficiency was different among the major HIV-1 genotypes,and ranked in descending order:CRF07＿BC(7.03y)>CRF55＿01B(5.71y,P=0.014;adjusted HR=3.752,P=0.092)>CRF01＿AE(5.18y,P<0.001;adjusted HR=4.733,P=0.015).HIV-1 genotype,baseline viral load(adjusted HR=3.311,P=0.004)and CD4 count(adjusted HR=0.021,P=0.046)are three independent variables significantly associated with disease progression.6.ART led to 36.0%(27/75)and 52.1%(38/73)of the patients whose anti-HIV-1 antibody levels at 12-and 24-months post-ART were>75%lower than at pre-treatment,respectively.The reduction of anti-HIV antibodies correlated with the decline of HIV1 viral load with correlation coefficients in the range 0.556-0.848(P<0.001).However,no negative detection of anti-HIV antibody was observed at 24 months post-ART.The time from HIV-1 diagnosis to ART initiation and the baseline anti-HIV-1 antibody level were significantly associated with quick decline of HIV-1 specific antibody during ART.Conclusions1.A LISA for detection of antibody against SARS-CoV-2 nucleoprotein was developed and evaluated in this study,and demonstrated superior performance in discriminating COVID-19 patients and uninfected subjects,which is semi-quantitative and is suitable for detecting the specific antibody in a wide range of species including human and animals.2.The study have shed light on the viral kinetics and specific antibody response in COVID-19 patients and provide scientific evidence for the development of SARSCoV-2 specific serological assay and the use of convalescent plasma in treatment for COVID-19 patients3.The current study developed and evaluated a CRISPR-Cas12a-based multiplex allele-specific assay for rapid detection and identification of SARS-CoV-2 variant.The system has the potential to be quickly developed,continuously updated and easily implemented for screening of SARS-CoV-2 variants in resource-limited settings.This approach can be adapted for emerging mutations and implemented in laboratories already conducting SARS-CoV-2 nucleic-acid amplification tests using existing resources and extracted SARS-CoV-2 nucleic acid.4.The study confirmed the differential rates of immunodeficiency progression as a function of HIV-1 genotype.The impact of HIV-1 genotypes on HIV epidemics,patient management and prevention should be further investigated.5.ART-induced kinetics of anti-HIV antibody response was different among the subjects with acute or chronic HIV-1 infection.Decline of HIV-1 specific antibody but no seroreversion was documented in HIV-1-infetced patients with delayed ART initiation.