ABSTRACT
SARS-CoV-2 vaccination is known to induce antibodies that recognize also variants of concerns (VoCs) of the virus. However, epidemiological and laboratory evidences indicate that these antibodies have a reduced neutralization ability against VoCs. We studied binding and neutralizing antibodies against the Spike protein domains and subunits of the Wuhan-Hu-1 virus and its alpha, beta, delta VoCs and of seasonal betacoronaviruses (HKU1 and OC43) in a cohort of 31 health care workers prospectively followed post-vaccination with BNT162b2-Comirnaty. The study of sequential samples collected up to 64 days post-vaccination showed that serological assays measuring IgG against Wuhan-Hu-1 antigens were a poor proxy for VoC neutralization. In addition, in subjects who had asymptomatic or mild COVID-19 prior to vaccination, the loss of nAbs following disease could be rapid and accompanied by post-vaccination antibody levels similar to those of naïve vaccinees. Interestingly, in health care workers naïve for SARS-CoV-2 infection, vaccination induced a rapid and transient reactivation of pre-existing seasonal coronaviruses IgG responses that was associated with a subsequent reduced ability to neutralize alpha and beta VoCs.
Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Neutralizing , Antibodies, Viral , BNT162 Vaccine , COVID-19/prevention & control , COVID-19 Vaccines , Humans , Immunoglobulin G , Seasons , VaccinationABSTRACT
Antiretroviral therapy has been shown to reduce rates of congenital CMV infection. Little information is available on the possible impact of antiretroviral therapy on postnatal breastfeeding-associated CMV infection acquisition. A cohort of 89 HIV-infected mothers and their children was studied. Women received antiretroviral therapy from week 25 of gestation until 6 months postpartum or indefinitely if meeting the criteria for treatment. All women were evaluated for CMV IgG presence and CMV DNA in breast milk. Children were tested for CMV infection by either the presence of IgM or the presence of CMV DNA in plasma at 1, 6 and 12 months and by the presence of IgG at 24 months. All mothers had high titers of CMV DNA in breast milk (5.7 log at Month 1 and 5.1 log at Month 6). Cumulative CMV infection rates were 60.3 % at Month 6, 69 % at Month 12 and 96.4 % at Month 24. There was a significant negative correlation between the duration of antiretroviral treatment during pregnancy and levels of CMV DNA in breast milk at Month 1 (P = 0.033). There was a trend for a correlation between high titers of CMV DNA in breast milk at 6 months and CMV infection at 6 months (P = 0.069). In this cohort, more than 95 % of the children had acquired CMV infection by 2 years of age. Besides breastfeeding, which played a major role, also horizontal transmission between 1 and 2 years was certainly relevant in determining CMV infection acquisition.
Subject(s)
Breast Feeding , Cytomegalovirus Infections/pathology , Cytomegalovirus Infections/transmission , Disease Transmission, Infectious , HIV Infections/complications , Infectious Disease Transmission, Vertical , Adult , Anti-Retroviral Agents/therapeutic use , Antibodies, Viral/blood , Child, Preschool , Cytomegalovirus Infections/diagnosis , DNA, Viral/analysis , Female , HIV Infections/drug therapy , Humans , Immunoglobulin G/blood , Infant , Infant, Newborn , Male , Milk, Human/virology , Pregnancy , Pregnancy Complications/drug therapy , Young AdultABSTRACT
There is limited information on the variations of HIV-1 DNA mutation profile in reverse transcriptase (RT) and protease (PR) genes during suppressive antiretroviral treatment (plasma HIV-1 RNA continuously <50 copies/ml) with raltegravir (RAL)-based regimens in patients with baseline RT/PR resistant HIV. Twelve multidrug resistant (RT: 12/12, PR: 8/12) HIV-infected patients were followed during effectively suppressive RAL-based therapy. Total and integrated HIV-1 DNA were assessed by real time PCR at baseline and every 6 months. Ultrasensitive (threshold: 2.5 copies/ml) plasma HIV-1 RNA and genotypic analysis of RT and PR in proviral DNA were performed at baseline and at 24 months. Half of the patients had full viral suppression (plasma HIV-RNA < 2.5 copies/ml) at month 12. Total HIV-1 DNA declined significantly after 12 months of therapy (from 249.2 to 145.7 copies/106 cells, P = 0.023), and remained stable until 24 months, when total HIV-1 DNA levels raised, concomitantly with a less stringent suppression of HIV-1 RNA (81.8% of patients with >2.5 copies/ml). Integrated HIV-1 DNA did not show fluctuations during the study period. Sequencing of the PR and RT regions from HIV-1 DNA revealed changes in the resistance mutation profile in five patients. Total HIV-1 DNA declined after the introduction of RAL-based therapy, with a rebound after 2 years. No changes were observed in levels of integrated DNA, suggesting limited effect on archived HIV. The RT and PR sequence changes in archived HIV-1 DNA suggest that variation of the mutation profile can occur even in the absence of detectable HIV-1 RNA. J. Med. Virol. 88:2115-2124, 2016. © 2016 Wiley Periodicals, Inc.
Subject(s)
Anti-HIV Agents/therapeutic use , DNA, Viral/genetics , Drug Resistance, Multiple, Viral , HIV Protease/genetics , HIV Reverse Transcriptase/genetics , HIV-1/enzymology , HIV-1/genetics , Raltegravir Potassium/therapeutic use , Adult , Anti-HIV Agents/adverse effects , Antiretroviral Therapy, Highly Active , Female , Genetic Variation , Genotype , HIV-1/physiology , Humans , Male , Middle Aged , Mutation , RNA, Viral/blood , RNA, Viral/genetics , Raltegravir Potassium/adverse effects , Viral LoadABSTRACT
BACKGROUND: Coinfection with the hepatitis viruses is common in the HIV population in sub-Saharan Africa. The aim of this study was to assess, in a cohort of HIV-infected pregnant women receiving antiretroviral drugs (ARVs), the prevalence of HBV and HCV infections and to determine the impact of these infections on the occurrence of liver toxicity and on the viro-immunological response. METHODS: Women were screened for HBsAg and HCV-RNA before starting, at week 25 of gestational age, an antiretroviral regimen consisting of lamivudine and nevirapine plus either stavudine or zidovudine. Women with CD4+ < 350/mm3 continued ARVs indefinitely, while the other women interrupted treatment 6 months postpartum (end of breastfeeding period). Both groups were followed for 2 years after delivery. Liver function was monitored by alanine aminotransferase (ALT) measurement. The Cox proportional hazards model was used to identify factors associated with the emergence of liver toxicity. RESULTS: A total of 28 women out of the 309 enrolled in the study (9.1%) were coinfected with HBV (n. 27), or HCV (n. 1). During follow-up 125 women (40.4%) developed a grade ≥ 1 ALT elevation, 28 (9.1%) a grade ≥ 2 and 6 (1.9%) an elevation defining grade 3 toxicity. In a multivariate model including age, baseline CD4+ count and hemoglobin level, the presence of either HBV or HCV infection was significantly associated with the development of an ALT increase of any grade (P = 0.035). Moderate or severe liver laboratory toxicity (grade ≥ 2) was more frequent among women with baseline CD4+ > 250/mm3 (P = 0.030). In HBV-infected women a baseline HBV-DNA level above 10,000 IU/ml was significantly associated to the development of liver toxicity of grade ≥ 1 (P = 0.040). Coinfections had no impact on the immunological and virological response to antiretroviral drugs up to 2 years after delivery. CONCLUSIONS: In this cohort of nevirapine-treated women the presence of HBV or HCV was associated only to the development of mild liver toxicity, while the occurrence of moderate or severe hepatoxicity was correlated to a baseline CD4+ count > 250/mm3. No statistically significant effect of the coinfections was observed on the efficacy of antiretroviral therapy.
Subject(s)
Anti-HIV Agents/therapeutic use , Chemical and Drug Induced Liver Injury/virology , Coinfection/epidemiology , HIV Infections/drug therapy , Hepatitis B/physiopathology , Hepatitis C/physiopathology , Nevirapine/therapeutic use , Pregnancy Complications, Infectious/drug therapy , Pregnancy Complications, Infectious/virology , Adult , Anti-HIV Agents/adverse effects , Coinfection/drug therapy , Coinfection/virology , Female , HIV Infections/transmission , HIV Infections/virology , Hepatitis B/virology , Hepatitis C/virology , Humans , Infectious Disease Transmission, Vertical/prevention & control , Nevirapine/adverse effects , Pregnancy , Young AdultABSTRACT
In sub-Saharan Africa the great majority of infants acquire Cytomegalovirus (CMV) infection within the first year of life. Maternal long-term antiretroviral therapy (ART) has been suggested to reduce the rate of CMV acquisition in HIV-exposed infants. In the present study serum samples collected at 6 months of age from HIV-exposed and HIV-unexposed infants were analyzed for the presence of CMV DNA (with CMV positivity defined by levels of CMV DNA > 1000 UI/ml). Twenty out of 58 (34.5%) infants had CMV DNA > 1000 UI/ml. There was no difference in the prevalence of CMV viremia between HIV-exposed and -unexposed infants [33.3% (15/45) vs 38.5% (5/13), respectively, P = 0.488]. In the HIV-exposed group, mothers of CMV-negative infants had received a longer antiretroviral treatment before delivery in comparison to mothers of CMV-positive infants (28 vs 3 months, P = 0.187). No differences in weights and lengths at birth, and at 1, 6 and 12 months were observed between CMV-positive and CMV-negative infants. In this study, the prevalence of CMV viremia at six months of age was high in infants born to HIV-positive mothers receiving long-term ART, similar to that of HIV-unexposed infants. Considering the possible relevant impact of CMV on infant health, strategies for containment of the infection should be explored.
Subject(s)
Cytomegalovirus Infections , HIV Infections , Pregnancy Complications, Infectious , Infant, Newborn , Female , Humans , Infant , Pregnancy , Cytomegalovirus , Viremia/drug therapy , Malawi/epidemiology , Infectious Disease Transmission, Vertical , Cytomegalovirus Infections/epidemiology , HIV Infections/drug therapy , HIV Infections/epidemiology , Pregnancy Complications, Infectious/drug therapy , Pregnancy Complications, Infectious/epidemiologyABSTRACT
BACKGROUND: The evaluation of seroprotection rates against vaccine-preventable infectious diseases allows for the identification of risk populations. HIV-exposed infants, even if not infected with HIV, have higher morbidity and mortality in comparison to unexposed counterparts. The aim of this study was to compare the specific IgG levels against Haemophilus influenzae type-B (HiB), Hepatitis-B (HBV), and Streptococcus pneumoniae (Spn) in two groups of infants (HIV-exposed and HIV-unexposed) living in Malawi. METHODS: Blood samples from 62 infants, 49 HIV-exposed, uninfected (HEU), and born to women living with HIV and 13 HIV-unexposed and uninfected (HUU), were collected at 6 months, and specific IgG levels were determined using ELISA tests. RESULTS: The antibody levels against HiB, HBV, and Spn were similar in the two groups. At six months, all HUU infants and 81.6% of HEU infants showed seroprotective levels against HiB, while a percentage of protection varying from 80.6 to 84.6% was observed for HBV and Spn regardless of HIV exposure. Only 59.2% of HEU and 69.2% of HUU infants showed antibody protection against all three pathogens. CONCLUSIONS: These results indicate similar rates of seroprotection among HEU and HUU infants but also suggest that a consistent fraction of infants received incomplete vaccinations. Strategies to enforce participation in immunization programs in Malawi should be a health priority.
ABSTRACT
Several COVID-19 vaccine strategies utilizing new formulations for the induction of neutralizing antibodies (nAbs) and T cell immunity are still under evaluation in preclinical and clinical studies. Here we used Simian Immunodeficiency Virus (SIV)-based integrase defective lentiviral vector (IDLV) delivering different conformations of membrane-tethered Spike protein in the mouse immunogenicity model, with the aim of inducing persistent nAbs against multiple SARS-CoV-2 variants of concern (VoC). Spike modifications included prefusion-stabilizing double proline (2P) substitutions, mutations at the furin cleavage site (FCS), D614G mutation and truncation of the cytoplasmic tail (delta21) of ancestral and Beta (B.1.351) Spike, the latter mutation to markedly improve IDLV membrane-tethering. BALB/c mice were injected once with IDLV delivering the different forms of Spike or the recombinant trimeric Spike protein with 2P substitutions and FCS mutations in association with a squalene-based adjuvant. Anti-receptor binding domain (RBD) binding Abs, nAbs and T cell responses were detected up to six months from a single immunization with escalating doses of vaccines in all mice, but with different levels and kinetics. Results indicated that IDLV delivering the Spike protein with all the combined modifications, outperformed the other candidates in terms of T cell immunity and level of both binding Abs and nAbs soon after the single immunization and persistence over time, showing the best capacity to neutralize all formerly circulating VoC Alpha, Beta, Gamma and Delta. Although present, the lowest response was detected against Omicron variants (BA.1, BA.2 and BA.4/5), suggesting that the magnitude of immune evasion may be related to the higher genetic distance of Omicron as indicated by increased number of amino acid substitutions in Spike acquired during virus evolution.
Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Animals , Humans , Mice , Spike Glycoprotein, Coronavirus/genetics , Integrases , COVID-19 Vaccines , SARS-CoV-2/genetics , Antibodies, Neutralizing , Disease Models, Animal , Mice, Inbred BALB C , ImmunityABSTRACT
OBJECTIVES: To evaluate the evolution of HIV-1 coreceptor tropism in proviral DNA of patients during maraviroc-based therapy. METHODS: Fourteen heavily high active antiretroviral therapy (HAART)-treated patients with a CCR5 Trofile profile were monitored over a 24 month period from the start of maraviroc therapy. Whole-blood samples were obtained at different timepoints, and coreceptor tropism was determined for proviral DNA from the V3-loop region sequence using the Geno2Pheno algorithm [false positive rate (FPR): 20%]. RESULTS: At the start of maraviroc treatment, 13/14 patients were viraemic (median: 4.33 log copies/mL). Concordance in R5 tropism (R5/R5) was observed between circulating HIV-RNA (Trofile) and HIV-DNA provirus in 10/14 patients (median FPR = 54.0%), while 4 patients showed a CXCR4-tropic R5/X4 variant in their provirus (FPR: 5.8%, 5.7%, 16.6% and 1.1%, respectively). All R5/R5 patients showed a stable HIV-1 DNA coreceptor usage. Two out of four R5/X4 patients showed a tropism shift in their archived provirus and, after 6 months a prevalence of R5-tropic virus was detected in DNA. The other two R5/X4 patients harboured the 11/25 genotype, and maintained X4 tropism in provirus during the study. Virological response did not reveal differences in RNA decay and CD4+ cell recovery in patients with discordant tropism. CONCLUSIONS: A relatively good correlation between RNA and DNA tropism was observed at baseline. Proviral DNA tropism remained stable over 24 months of maraviroc-based therapy, indicating that determination of proviral DNA V3 sequence could be used in tropism prediction in clinical practice. The data also confirm the importance of the 11/25 rule in predicting viral tropism.
Subject(s)
Anti-HIV Agents/administration & dosage , Cyclohexanes/administration & dosage , Evolution, Molecular , HIV Infections/drug therapy , HIV-1/physiology , Proviruses/physiology , Triazoles/administration & dosage , Viral Tropism , Adult , Anti-HIV Agents/pharmacology , Cyclohexanes/pharmacology , DNA, Viral/genetics , Female , HIV Envelope Protein gp120/genetics , HIV Infections/virology , HIV-1/genetics , Humans , Male , Maraviroc , Middle Aged , Proviruses/genetics , Selection, Genetic , Triazoles/pharmacologyABSTRACT
Integrase Defective Lentiviral Vectors (IDLVs) represent an attractive vaccine platform for delivering HIV-1 antigens, given their ability to induce specific and persistent immune responses in both mice and non-human primates (NHPs). Recent advances in HIV-1 immunogen design demonstrated that native-like HIV-1 Envelope (Env) trimers that mimic the structure of virion-associated Env induce neutralization breadth in rabbits and macaques. Here, we describe the development of an IDLV-based HIV-1 vaccine expressing either soluble ConSOSL.UFO.664 or membrane-tethered ConSOSL.UFO.750 native-like Env immunogens with enhanced bNAb epitopes exposure. We show that IDLV can be pseudotyped with properly folded membrane-tethered native-like UFO.750 trimers. After a single IDLV injection in BALB/c mice, IDLV-UFO.750 induced a faster humoral kinetic as well as higher levels of anti-Env IgG compared to IDLV-UFO.664. IDLV-UFO.750 vaccinated cynomolgus macaques developed unusually long-lasting anti-Env IgG antibodies, as underlined by their remarkable half-life both after priming and boost with IDLV. After boosting with recombinant ConM SOSIP.v7 protein, two animals developed neutralization activity against the autologous tier 1B ConS virus mediated by V1/V2 and V3 glycan sites responses. By combining the possibility to display stabilized trimeric Env on the vector particles with the ability to induce sustained humoral responses, IDLVs represent an appropriate strategy for delivering rationally designed antigens to progress towards an effective HIV-1 vaccine.
ABSTRACT
OBJECTIVES: Simplified methods for virological monitoring in resource-limited settings are increasingly needed. We evaluated the performance of the VERSANT(®) HIV-1 RNA (kPCR) assay for the determination of HIV-1 viral load from dried blood spots (DBS). Assay sensitivity and correlation with plasma quantification values were assessed. METHODS: A total of 98 DBS were prepared from fresh blood samples of HIV-infected patients. DBS were kept at room temperature for 6 weeks or 7 months before processing while the corresponding plasma samples were stored at -80°C. DBS were first pre-treated in a special DBS buffer. The DBS extracts and the plasma samples were then purified and amplified using the VERSANT assay reagents. RESULTS: In the first series of tests, performed after 6 weeks of storage, there was good correlation between quantification of viral load in plasma and in DBS (r = 0.95, P < 0.001). The detection rate in DBS was 100% when plasma levels were >1000 copies/mL. The sensitivity and specificity of the DBS assay were 88.2% [95% confidence interval (CI) 79.4-93.6] and 69.2% (95% CI 42.0-87.4), respectively. Using the 5000 copies/mL threshold (defining virological failure in resource-limited settings), both positive and negative predictive values were high (95.2% and 87.5%, respectively). After 7 months of storage there was a modest decrease in the detection rate and less significant correlations for samples with HIV-RNA <5000 copies/mL. CONCLUSIONS: Quantification of HIV-RNA from DBS by the VERSANT automated sample preparation and detection method can be used to diagnose virological failure in HIV-positive patients.
Subject(s)
Blood/virology , Desiccation/methods , HIV Infections/virology , HIV-1/isolation & purification , Molecular Diagnostic Techniques/methods , Specimen Handling/methods , Viral Load , Automation/methods , Humans , RNA, Viral/blood , Reagent Kits, Diagnostic , Sensitivity and Specificity , Virology/methodsABSTRACT
Integrase-defective lentiviral vectors (IDLVs) represent an attractive platform for vaccine development as a result of the ability to induce persistent humoral- and cellular-mediated immune responses against the encoded transgene. Compared with the parental integrating vector, the main advantages for using IDLV are the reduced hazard of insertional mutagenesis and the decreased risk for vector mobilization by wild-type viruses. Here we report on the development and use in the mouse immunogenicity model of simian immunodeficiency virus (SIV)-based IDLV containing a long deletion in the U3 region and with the 3' polypurine tract (PPT) removed from the transfer vector for improving safety and/or efficacy. Results show that a safer extended deletion of U3 sequences did not modify integrase-mediated or -independent integration efficiency. Interestingly, 3' PPT deletion impaired integrase-mediated integration but did not reduce illegitimate, integrase-independent integration efficiency, contrary to what was previously reported in the HIV system. Importantly, although the extended deletion in the U3 did not affect expression or immunogenicity from IDLV, deletion of 3' PPT considerably reduced both expression and immunogenicity of IDLV.
ABSTRACT
CONTEXT: Demonstrating the ability to mount a neutralizing antibody response to SARS-CoV-2 in the presence of diabetes is crucial to understand COVID-19 pathogenesis, reinfection potential, and vaccine development. OBJECTIVE: The aim of this study was to characterize the kinetics and durability of neutralizing antibody (Nab) response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the presence of hyperglycemia. METHODS: Using a lentiviral vector-based SARS-CoV-2 neutralization assay to measure Nabs, we characterized 150 patients randomly selected from a cohort of 509 patients with confirmed COVID-19 pneumonia. We analyzed Nab response according to the presence of diabetes or hyperglycemia, at the time of hospitalization and during the postdischarge follow-up: 1-, 3-, and 6-month outpatient visits. RESULTS: Among 150 randomly selected patients 40 (26.6%) had diabetes. Diabetes (hazard ratio [HR] 8.9, P < .001), glucose levels (HR 1.25 × 1.1 mmol/L, P < .001), and glucose variability (HR 1.17 × 0.6 mmol/L, P < .001) were independently associated with an increased risk of mortality. The neutralizing activity of SARS-CoV-2 antibodies in patients with diabetes was superimposable, as for kinetics and extent, to that of patients without diabetes. It was similar across glucose levels and correlated with the humoral response against the SARS-CoV-2 spike protein. Positivity for Nabs at the time of hospital admission conferred protection on mortality, both in the presence (HR 0.28, P = .046) or absence of diabetes (HR 0.26, P = .030). The longevity of the Nab response was not affected by diabetes. CONCLUSION: Diabetes and hyperglycemia do not affect the kinetics and durability of the neutralizing antibody response to SARS-CoV-2. These findings provide the rational to include patients with diabetes in the early phase of the vaccination campaign against SARS-CoV-2.
Subject(s)
Antibodies, Neutralizing/immunology , COVID-19/immunology , Diabetes Complications/immunology , Pneumonia/immunology , COVID-19/complications , Diabetes Complications/virology , Female , Humans , Male , Pneumonia/complicationsABSTRACT
Integrase-defective lentiviral vectors (IDLVs) have been used as a safe and efficient delivery system in several immunization protocols in murine and non-human primate preclinical models as well as in recent clinical trials. In this work, we validated in preclinical murine models our vaccine platform based on IDLVs as delivery system for cancer immunotherapy. To evaluate the anti-tumor activity of our vaccine strategy we generated IDLV delivering ovalbumin (OVA) as a non-self-model antigen and TRP2 as a self-tumor associated antigen (TAA) of melanoma. Results demonstrated the ability of IDLVs to eradicate and/or controlling tumor growth after a single immunization in preventive and therapeutic approaches, using lymphoma and melanoma expressing OVA. Importantly, LV-TRP2 but not IDLV-TRP2 was able to break tolerance efficiently and prevent tumor growth of B16F10 melanoma cells. In order to improve the IDLV efficacy, the human homologue of murine TRP2 was used, showing the ability to break tolerance and control the tumor growth. These results validate the use of IDLV for cancer therapy.
Subject(s)
Cancer Vaccines/administration & dosage , Genetic Vectors/genetics , Immunotherapy , Integrases/metabolism , Lentivirus/genetics , Melanoma/immunology , Melanoma/therapy , Animals , Cancer Vaccines/genetics , Cancer Vaccines/immunology , Genetic Vectors/metabolism , Humans , Integrases/genetics , Intramolecular Oxidoreductases/administration & dosage , Intramolecular Oxidoreductases/genetics , Intramolecular Oxidoreductases/immunology , Lentivirus/enzymology , Lentivirus/metabolism , Male , Melanoma/genetics , Mice , Mice, Inbred C57BL , VaccinationABSTRACT
Understanding how antibody responses to SARS-CoV-2 evolve during infection may provide important insight into therapeutic approaches and vaccination for COVID-19. Here we profile the antibody responses of 162 COVID-19 symptomatic patients in the COVID-BioB cohort followed longitudinally for up to eight months from symptom onset to find SARS-CoV-2 neutralization, as well as antibodies either recognizing SARS-CoV-2 spike antigens and nucleoprotein, or specific for S2 antigen of seasonal beta-coronaviruses and hemagglutinin of the H1N1 flu virus. The presence of neutralizing antibodies within the first weeks from symptoms onset correlates with time to a negative swab result (p = 0.002), while the lack of neutralizing capacity correlates with an increased risk of a fatal outcome (p = 0.008). Neutralizing antibody titers progressively drop after 5-8 weeks but are still detectable up to 8 months in the majority of recovered patients regardless of age or co-morbidities, with IgG to spike antigens providing the best correlate of neutralization. Antibody responses to seasonal coronaviruses are temporarily boosted, and parallel those to SARS-CoV-2 without dampening the specific response or worsening disease progression. Our results thus suggest compromised immune responses to the SARS-CoV-2 spike to be a major trait of COVID-19 patients with critical conditions, and thereby inform on the planning of COVID-19 patient care and therapy prioritization.
Subject(s)
Antibodies, Neutralizing/immunology , COVID-19/immunology , COVID-19/mortality , SARS-CoV-2/immunology , Aged , Antibodies, Viral/immunology , Antibody Formation , Betacoronavirus/immunology , COVID-19/virology , Female , Humans , Immunoglobulin G/immunology , Kinetics , Longitudinal Studies , Male , Middle Aged , Neutralization Tests , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/immunology , Survival RateABSTRACT
Antibodies targeting Receptor Binding Domain (RBD) of SARS-CoV-2 have been suggested to account for the majority of neutralizing activity in COVID-19 convalescent sera and several neutralizing antibodies (nAbs) have been isolated, characterized and proposed as emergency therapeutics in the form of monoclonal antibodies (mAbs). However, SARS-CoV-2 variants are rapidly spreading worldwide from the sites of initial identification. The variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.167.2 (Delta) showed mutations in the SARS-CoV-2 spike protein potentially able to cause escape from nAb responses with a consequent reduction of efficacy of vaccines and mAbs-based therapy. We produced the recombinant RBD (rRBD) of SARS-CoV-2 spike glycoprotein from the Wuhan-Hu 1 reference sequence in a mammalian system, for mice immunization to isolate new mAbs with neutralizing activity. Here we describe four mAbs that were able to bind the rRBD in Enzyme-Linked Immunosorbent Assay and the transmembrane full-length spike protein expressed in HEK293T cells by flow cytometry assay. Moreover, the mAbs recognized the RBD in supernatants of SARS-CoV-2 infected VERO E6 cells by Western Blot under non-reducing condition or in supernatants of cells infected with lentivirus pseudotyped for spike protein, by immunoprecipitation assay. Three out of four mAbs lost their binding efficiency to completely N-deglycosylated rRBD and none was able to bind the same recombinant protein expressed in Escherichia coli, suggesting that the epitopes recognized by three mAbs are generated by the conformational structure of the glycosylated native protein. Of particular relevance, three mAbs were able to inhibit Wuhan SARS-CoV-2 infection of VERO E6 cells in a plaque-reduction neutralization test and the Wuhan SARS-CoV-2 as well as the Alpha, Beta, Gamma and Delta VOC in a pseudoviruses-based neutralization test. These mAbs represent important additional tools for diagnosis and therapy of COVID-19 and may contribute to the understanding of the functional structure of SARS-CoV-2 RBD.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , Epitopes/immunology , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/genetics , Animals , Binding Sites, Antibody/immunology , Cell Line, Tumor , Chlorocebus aethiops , Female , Glycosylation , HEK293 Cells , Humans , Mice, Inbred BALB C , Neutralization Tests , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Vero Cells , COVID-19 Drug TreatmentABSTRACT
INTRODUCTION: Microbial translocation (MT) markers are indicators of HIV-related immune activation, but reference values are mostly derived from European or North American populations and could be substantially different in populations living in developing countries. Here we evaluate possible differences in MT markers levels in HIV+ pregnant women of different geographical provenance. METHODOLOGY: This study is nested within an observational study of pregnant women with HIV in Italy. Women were dichotomized on the basis of provenance in two groups of European (n = 14) and African (n = 26) origin. Soluble CD14, lipopolysaccharide-binding protein (LBP) and intestinal-fatty acid binding protein (I-FABP) were measured in plasma samples collected between the first and second trimester of pregnancy. RESULTS: Demographic and viroimmunological characteristics were similar between groups, although European women were more commonly smokers and HCV-coinfected. Irrespective of origin, LBP plasma levels were positively correlated with I-FABP (r = 0.467, p = 0.004) and sCD14 levels (r = 0.312 p = 0.060). Significantly higher levels of sCD14 (1885 vs. 1208 ng/mL, p = 0.005) LBP (28.5 vs. 25.3 µg/mL, p = 0.050) and I-FABP (573.4 vs. 358.2 pg/mL, p = 0.002) were observed in European compared with African women. A multivariable linear regression analysis, adjusted for smoking and HCV coinfection confirmed the association between sCD14 levels and women provenance (p = 0.03). CONCLUSIONS: Our observations indicate significant differences in soluble markers among women of different provenance. In the design and analysis of studies evaluating MT markers, population-specific reference values should be considered.
Subject(s)
Carrier Proteins/blood , Fatty Acid-Binding Proteins/blood , HIV Infections/blood , Lipopolysaccharide Receptors/blood , Membrane Glycoproteins/blood , Pregnancy Complications, Infectious/blood , Acute-Phase Proteins , Adult , Africa , Biomarkers/blood , Coinfection/blood , Coinfection/complications , Coinfection/microbiology , Cytomegalovirus Infections/blood , Cytomegalovirus Infections/complications , Cytomegalovirus Infections/microbiology , Europe , Female , HIV Infections/complications , HIV Infections/microbiology , HIV-1 , Hepatitis, Chronic/blood , Hepatitis, Chronic/complications , Hepatitis, Chronic/microbiology , Humans , Plasma/chemistry , Pregnancy , Pregnancy Complications, Infectious/microbiology , Pregnancy Complications, Infectious/virology , Pregnancy Trimester, First , Pregnancy Trimester, SecondABSTRACT
Cellular immune responses play a fundamental role in controlling viral replication and AIDS progression in human immunodeficiency virus (HIV)-infected subjects and in simian immunodeficiency virus (SIV)-infected macaques. Integrase defective lentiviral vector (IDLV) represents a promising vaccine candidate, inducing functional and durable immune responses in mice and non-human primates. Here, we designed HIV- and SIV-based IDLVs to express the HIVACAT T cell immunogen (HTI), a mosaic antigen designed to cover vulnerable sites in HIV-1 Gag, Pol, Vif, and Nef. We observed that HTI expression during lentiviral vector production interfered profoundly with IDLV particles release because of sequestration of both HIV- and SIV-Gag proteins in the cytoplasm of the vector-producing cells. However, modifications in IDLV design and vector production procedures greatly improved recovery of both HIV- and SIV-based IDLV-HTI. Immunization experiments in BALB/c mice showed that both IDLVs elicited HTI-specific T cell responses. However, immunization with HIV-based IDLV elicited also a T cell response toward exogenous HIV proteins in IDLV particles, suggesting that SIV-based IDLV may be a preferable platform to assess the induction of transgene-specific immune responses against rationally designed HIV structural antigens. These data support the further evaluation of IDLV as an effective platform of T cell immunogens for the development of an effective HIV vaccine.
ABSTRACT
BACKGROUND: Virtually all HIV-infected women in sub-Saharan Africa have evidence of Cytomegalovirus (CMV) infection and levels of specific anti-CMV IgG have been suggested to represent more intense reactivation of subclinical infection. Studies have also shown direct influence of CMV on lymphocytes. OBJECTIVE: The aim of this study was to determine if levels of anti-CMV specific antibodies could impact on the immunological response to antiretroviral treatment (ART) in HIV-infected pregnant women. STUDY DESIGN: CMV-specific IgG were measured in HIV-infected pregnant women at 26 weeks of gestation (before ART initiation). Women received ART until 6 months postpartum or indefinitely according to local guidelines at the time of the study. Immunological and virological responses were assessed 6 months and 24 months after delivery. RESULTS: A total of 81 women were studied. At baseline high levels (above the median) of specific IgG were associated to a low CD4+ cell count (P<0.001), a high viral load (P=0.003), and to an older age (P=0.051). In a multivariate model adjusting for baseline CD4+ count, baseline viral load and age, the presence of low levels of CMV IgG was the only independent predictor of a a CD4+ count above 500/mm3 24 months after delivery among women on continuous therapy. CONCLUSIONS: In this cohort, levels of CVM IgG had a significant influence on the immunological response to ART, adding information to the known impact of CMV infection in the HIV-positive population, and underlining the need of new strategies to contain the infection.
Subject(s)
Anti-Retroviral Agents/therapeutic use , Antibodies, Viral/blood , CD4-Positive T-Lymphocytes/immunology , Cytomegalovirus Infections/immunology , HIV Infections/immunology , Immunoglobulin G/blood , Pregnancy Complications, Infectious/immunology , Adult , Africa South of the Sahara , CD4 Lymphocyte Count , Coinfection/immunology , Coinfection/virology , Cytomegalovirus Infections/complications , Female , HIV Infections/complications , HIV Infections/drug therapy , Humans , Immunity, Cellular , Immunity, Humoral , Pregnancy , Pregnancy Complications, Infectious/virology , Viral Load , Young AdultABSTRACT
The objective of this study was to determine the virological response and the possible emergence of drug resistance at 1 and 2 years postpartum in HIV-positive pregnant women enrolled under the Option B approach and meeting the criteria for treatment. In the study, women with baseline CD4(+) <350/mm(3) received a combination of stavudine, lamivudine, and nevirapine during pregnancy (from week 25 of gestation) and continued it indefinitely after delivery. HIV-RNA was measured at 12 and 24 months postpartum. Drug resistance mutations were assessed in those with HIV-RNA >50 copies/ml. Baseline resistance mutations were assessed in the entire cohort. A total of 107 women were studied. At baseline, resistance mutations were seen in 6.6% of the women. At 12 months, 26.7% of the women had >50 copies/ml and among them 12.9% had virological failure (HIV-RNA >1,000 copies/ml). At 24 months, detectable HIV-RNA was seen in 28.3% of the women and virological failure in 10.1% of the women. Resistance mutations (mainly non-nucleoside reverse transcriptase inhibitors mutations) were seen in 40% of the women with detectable HIV-RNA. Baseline mutations did not correlate with virological failure or the emergence of resistance at later time points. Virological failure 2 years postpartum and emergence of resistance were rare in this cohort of HIV-infected women. These findings are reassuring in the light of the new strategies for the prevention of mother-to-child HIV transmission, recommending life-long antiretroviral therapy administration.
Subject(s)
Anti-HIV Agents/therapeutic use , HIV Infections/drug therapy , HIV-1/drug effects , Infectious Disease Transmission, Vertical/prevention & control , Lamivudine/therapeutic use , Nevirapine/therapeutic use , Stavudine/therapeutic use , Adult , Antiretroviral Therapy, Highly Active , CD4 Lymphocyte Count , Drug Resistance, Viral/genetics , Female , Gestational Age , HIV Infections/immunology , HIV Infections/virology , HIV-1/genetics , HIV-1/growth & development , Humans , Malawi , Mutation , Postpartum Period , Pregnancy , RNA, Viral/antagonists & inhibitors , RNA, Viral/biosynthesis , Viral Load/drug effectsABSTRACT
OBJECTIVE: To determine factors associated with < 2.5 copies/ml plasma HIV RNA in subjects treated with highly active antiretroviral therapy (HAART) and with viraemia < 50 copies/ml. DESIGN: Cross-sectional analysis of 84 HIV-positive patients taking HAART with plasma HIV RNA < 50 copies/ml for at least 6 months and no history of virological failure. METHODS: Current HAART therapy was based on a non-nucleoside reverse transcriptase inhibitor (NNRTI) in 66%, a protease inhibitor in 26% and nucleoside reverse transcriptase inhibitors in 7%. Viraemia levels were measured using a modified ultrasensitive Roche Amplicor HIV-1 Monitor test able to quantify plasma HIV RNA to a lower limit of 2.5 copies /ml; proviral DNA was measured with a real-time polymerase chain reaction assay. Analysis of variance and multiple logistic regression analysis were utilized to test for associations between residual replication and other variables. RESULTS: Residual HIV viraemia > 2.5 copies/ml was found in 50% of subjects; 94% of subjects had detectable proviral DNA (>or= 20 copies/10(6) peripheral blood mononuclear cells) and 21% had archived mutations. Usage of a NNRTI-based HAART was the only independent predictor of viral suppression below the cut-off value of the modified ultrasensitive assay. CONCLUSIONS: In our population, NNRTI-based HAART seems to have a stronger impact on residual replication than protease inhibitor-based HAART. This finding may be considered in therapeutic decisions such as the choice of initial HAART regimen and the interruption or simplification of treatment.