ABSTRACT
Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-191,2. A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-193. Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches.
Subject(s)
Antibodies, Bispecific/immunology , Antibodies, Neutralizing/immunology , COVID-19/immunology , COVID-19/virology , Immunoglobulin G/immunology , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Bispecific/therapeutic use , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/therapeutic use , Body Weight , COVID-19/prevention & control , Dependovirus/genetics , Disease Models, Animal , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/immunology , Female , Humans , Immune Evasion/genetics , Mice , Mice, Inbred C57BL , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , COVID-19 Drug TreatmentABSTRACT
The emergence of Omicron lineages and descendent subvariants continues to present a severe threat to the effectiveness of vaccines and therapeutic antibodies. We have previously suggested that an insufficient mucosal immunoglobulin A (IgA) response induced by the mRNA vaccines is associated with a surge in breakthrough infections. Here, we further show that the intramuscular mRNA and/or inactivated vaccines cannot sufficiently boost the mucosal secretory IgA response in uninfected individuals, particularly against the Omicron variant. We thus engineered and characterized recombinant monomeric, dimeric, and secretory IgA1 antibodies derived from four neutralizing IgG monoclonal antibodies (mAbs 01A05, rmAb23, DXP-604, and XG014) targeting the receptor-binding domain of the spike protein. Compared to their parental IgG antibodies, dimeric and secretory IgA1 antibodies showed a higher neutralizing activity against different variants of concern (VOCs), in part due to an increased avidity. Importantly, the dimeric or secretory IgA1 form of the DXP-604 antibody significantly outperformed its parental IgG antibody, and neutralized the Omicron lineages BA.1, BA.2, and BA.4/5 with a 25- to 75-fold increase in potency. In human angiotensin converting enzyme 2 (ACE2) transgenic mice, a single intranasal dose of the dimeric IgA DXP-604 conferred prophylactic and therapeutic protection against Omicron BA.5. Thus, dimeric or secretory IgA delivered by nasal administration may potentially be exploited for the treatment and prevention of Omicron infection, thereby providing an alternative tool for combating immune evasion by the current circulating subvariants and, potentially, future VOCs.
Subject(s)
Antibodies, Monoclonal , Immunoglobulin A, Secretory , Animals , Mice , Humans , Immunoglobulin G , Immunoglobulin A , Administration, Intranasal , Mice, TransgenicABSTRACT
Monkeypox virus (MPXV) is a zoonotic disease endemic in the rainforest countries of Central and West Africa. Understanding the immune response in zoonosis is fundamental to prevent and contrast viral spreading. MPXV is a close relative of Variola (smallpox) virus and vaccination with vaccinia virus gives approximatively 85% of protection against MPXV. With the emergence of the recent MPXV outbreak, JYNNEOS vaccine has been proposed to individuals at high-risk of exposure. Comparative data on MPXV immune response in vaccinated or infected subjects are still limited. Here we set-up an immunofluorescence method for the evaluation of humoral response elicited by natural infection and healthy vaccinated subjects, including historically smallpox-vaccinated individuals and newly vaccinated subjects. Neutralization assay was also included, and in vaccinated subjects, cell-mediated response was evaluated. We observed that the natural infection produces a strong immune response that can control the disease. In naïve subjects, a second dose boosts the serological response to levels similar to those of the MPXV patients. Last, smallpox-vaccinated controls retain a degree of protection, even after years from vaccination, most visible in the t-cellular response.
Subject(s)
Mpox (monkeypox) , Smallpox , Humans , Monkeypox virus , Smallpox/prevention & control , Mpox (monkeypox)/epidemiology , Mpox (monkeypox)/prevention & control , Vaccinia virus , ImmunityABSTRACT
The role and durability of the immunogenicity of the BNT162b2 mRNA vaccine against severe acute respiratory virus 2 (SARS-CoV-2), in cancer patients one year after receiving the third dose have to be elucidated. We have prospectively evaluated the long-term immunogenicity of the third dose of the SARS-CoV-2 BNT162b2 mRNA vaccine in 55 patients undergoing active treatment. Neutralizing antibody (NT Ab) titers against Omicron variants and total anti-trimeric S IgG levels were measured one year after the third dose. Heparinized whole-blood samples were used for the assessment of the SARS-CoV-2 interferon-γ release assay (IGRA). Thirty-seven patients (67.3%) showed positive total anti-trimeric S IgG one year after the third dose. Looking at the T-cell response against the spike protein, the frequency of responder patients did not decrease significantly between six and twelve months after the third dose. Finally, less than 20% of cancer patients showed an undetectable NT Ab titer against BA.1 and BA.5 variants of concern (VOCs). Underlying therapies seem to not affect the magnitude or frequency of the immune response. Our work underlines the persistence of humoral and cellular immune responses against BNT162b2 in a cohort of cancer patients one year after receiving the third dose, regardless of the type of underlying therapy.
Subject(s)
COVID-19 , Neoplasms , Virus Diseases , Humans , BNT162 Vaccine , COVID-19 Vaccines , Follow-Up Studies , SARS-CoV-2 , COVID-19/prevention & control , Neoplasms/therapy , Antibodies, Neutralizing , Immunity , Immunoglobulin G , Antibodies, Viral , mRNA VaccinesABSTRACT
Optimal vaccines are needed for sustained suppression of SARS-CoV-2 and other novel coronaviruses. Here, we developed a recombinant type 5 adenovirus vector encoding the gene for the SARS-CoV-2 S1 subunit antigen (Ad5.SARS-CoV-2-S1) for COVID-19 immunization and evaluated its immunogenicity in mice. A single immunization with Ad5.SARS-CoV-2-S1 via S.C. injection or I.N delivery induced robust antibody and cellular immune responses. Vaccination elicited significant S1-specific IgG, IgG1, and IgG2a endpoint titers as early as 2 weeks, and the induced antibodies were long lasting. I.N. and S.C. administration of Ad5.SARS-CoV-2-S1 produced S1-specific GC B cells in cervical and axillary LNs, respectively. Moreover, I.N. and S.C. immunization evoked significantly greater antigen-specific T-cell responses compared to unimmunized control groups with indications that S.C. injection was more effective than I.N. delivery in eliciting cellular immune responses. Mice vaccinated by either route demonstrated significantly increased virus-specific neutralization antibodies on weeks 8 and 12 compared to control groups, as well as BM antibody forming cells (AFC), indicative of long-term immunity. Thus, this Ad5-vectored SARS-CoV-2 vaccine candidate showed promising immunogenicity following delivery to mice by S.C. and I.N. routes of administration, supporting the further development of Ad-based vaccines against COVID-19 and other infectious diseases for sustainable global immunization programs.
Subject(s)
COVID-19 Vaccines/immunology , COVID-19/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adenoviridae/genetics , Adenoviridae/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , B-Lymphocytes/immunology , Immunity, Cellular/immunology , Immunity, Humoral/immunology , Immunoglobulin G/immunology , Mice , Mice, Inbred BALB C , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , T-Lymphocytes/immunology , VaccinationABSTRACT
BACKGROUND: Novel SARS-CoV-2 variants of concern (VOC) Delta and Omicron are able to escape some monoclonal antibody therapies, making again COVID-19 convalescent plasma (CCP) a potential frontline treatment. STUDY DESIGN/METHODS: In this study, we investigated the kinetics of anti-SARS-CoV-2 neutralizing antibodies (nAbs) against VOCs Delta and Omicron in vaccine breakthrough infected plasma donors. Serum samples from 19 donors were collected at the time of plasma donation and tested for anti-SARS-CoV-2 nAbs (using live authentic VOC viral neutralization test) and IgG (Liaison® SARS-CoV-2 S1/S2 and Liaison® SARS-CoV-2 TrimericS IgG assays, DiaSorin). Measures were correlated with different variables, including the time between last vaccine dose and CCP donation, and time between SARS-COV-2 infection and CCP donation. RESULTS: nAb titers against VOC Delta and Omicron were directly related to the time interval since last vaccine dose to CCP donation, but inversely related to time since COVID19 breakthrough infection. DISCUSSION: SARS-CoV-2 breakthrough infection in vaccinated in donors boosts nAb titers against VOCs Delta and Omicron, but such titers decay shortly after infection. Therefore, CCP must be collected early after vaccine breakthrough infection.
Subject(s)
COVID-19 , Viral Vaccines , Antibodies, Neutralizing , Antibodies, Viral , Blood Donors , COVID-19/prevention & control , COVID-19/therapy , Humans , Immunization, Passive , Immunoglobulin G , Neutralization Tests , SARS-CoV-2 , COVID-19 SerotherapyABSTRACT
Siglec-15 is a conserved sialic acid-binding Ig-like lectin, which is expressed on osteoclasts. Deficiency of Siglec-15 leads to an impaired osteoclast development, resulting in a mild osteopetrotic phenotype. The role of Siglec-15 in arthritis is still largely unclear. To address this, we generated Siglec-15 knockout mice and analyzed them in a mouse arthritis model. We could show that Siglec-15 is directly involved in pathologic bone erosion in the K/BxN serum-transfer arthritis model. Histological analyses of joint destruction provided evidence for a significant reduction in bone erosion area and osteoclast numbers in Siglec-15-/- mice, whereas the inflammation area and cartilage destruction was comparable to wild-type mice. Thus, Siglec-15 on osteoclasts has a crucial function for bone erosion during arthritis. In addition, we generated a new monoclonal anti-Siglec-15 Ab to clarify its expression pattern on immune cells. Whereas this Ab demonstrated an almost exclusive Siglec-15 expression on murine osteoclasts and hardly any other expression on various other immune cell types, human Siglec-15 was more broadly expressed on human myeloid cells, including human osteoclasts. Taken together, our findings show a role of Siglec-15 as a regulator of pathologic bone resorption in arthritis and highlight its potential as a target for future therapies, as Siglec-15 blocking Abs are available.
Subject(s)
Arthritis, Rheumatoid/immunology , Bone Resorption/immunology , Immunoglobulins/metabolism , Membrane Proteins/metabolism , Osteoclasts/metabolism , Animals , Arthritis, Experimental/blood , Arthritis, Experimental/complications , Arthritis, Experimental/genetics , Arthritis, Experimental/immunology , Arthritis, Rheumatoid/blood , Arthritis, Rheumatoid/complications , Arthritis, Rheumatoid/genetics , Bone Resorption/pathology , Bone and Bones/immunology , Bone and Bones/pathology , Cells, Cultured , Female , Humans , Immunoglobulins/genetics , Leukocytes, Mononuclear , Male , Membrane Proteins/genetics , Mice , Mice, Knockout , Osteoclasts/immunology , Primary Cell CultureABSTRACT
BACKGROUND: Test the ability of Mirasol Pathogen Reduction Technology (PRT, Terumo BCT, Lakewood Co, USA) treatment with riboflavin and ultraviolet light (R + UV) in reducing SARS-CoV-2 infectivity while maintaining blood product quality. MATERIAL AND METHODS: SARS-CoV-2 strains were isolated and titrated to prepare cell free virus for plasma units infection. The units were then under treatment with Mirasol PRT. The infectious titers were determined before and after treatment with an in house microtitration assay on Vero E6 cells. Thirty-six plasma pool bags underwent PRT treatment. RESULTS: In all the experiments, the measured titer following riboflavin and UV treatment was below the limit of detection of microtitration assay for all the different SARS-CoV-2 strains. Despite the high copies number detected by RT-PCR for each viral strain after treatment, viruses were completely inactivated and not able to infect VERO E6 cells. CONCLUSION: Riboflavin and UV light treatment effectively reduced the virus titers of human plasma to the limit of detection in tissue culture, regardless of the strain. These data suggest that pathogen reduction in blood products highlight the safety of CP therapy procedures for critically ill COVID-19 patients, while maintaining blood product quality.
Subject(s)
COVID-19 Drug Treatment , SARS-CoV-2 , Humans , Riboflavin/pharmacology , Ultraviolet RaysABSTRACT
Letermovir (LMV) inhibits HCMV replication by binding to components of the HCMV-terminase complex showing a potential role in prevention of HCMV-related complications in allogenic hematopoietic stem cell transplant recipients (allo-HSCTRs). However, little is known about breakthrough HCMV infection and the relevance of HCMV DNAemia during prophylaxis. We reported the results of a multicenter prospective study involving five Italian centers in the management of HCMV DNAemia in 75 adult HCMV-seropositive allo-HSCTRs undergoing LMV prophylaxis. The aim of the present study was to characterize the presence of real HCMV reactivation during LMV prophylaxis. Then, the presence of circulating infectious HCMV particles was determined by virus isolation and degradation of free-floating viral DNA. This report provides the first evidence that during LMV prophylaxis the clinical relevance of HCMV DNAemia should be critically considered.
Subject(s)
Antiviral Agents , Cytomegalovirus , Acetates , Antiviral Agents/therapeutic use , Cytomegalovirus/genetics , DNA, Viral/genetics , Prospective Studies , Quinazolines , Stem CellsABSTRACT
BACKGROUND: Although many trials are currently investigating the safety and efficacy of convalescent plasma (CP) in critically ill COVID-19 patients, there is a paucity of ongoing and published studies evaluating the CP donors' side. This retrospective study reports the first Italian experience on CP donors' selection and donations. METHODS: Patients aged 18-68 years who had recovered from COVID-19 at least 2 weeks previously were recruited between March 18 and June 30, 2020 in a study protocol at the Italian hospitals of Pavia and Mantova. RESULTS: During the study period, 494 of 512 donors recruited were judged eligible and underwent 504 plasmapheresis procedures. Eighty-five percent (437/512) of the CP donors were males. The average time between symptom recovery and CP donation was 36.6 (±20.0) days. Four hundred and eighty-eight plasmapheresis procedures (96.8%) were concluded and each unit was divided into two subunits (total 976) with an average volume of 316.2 (±22.7) mL. Ninety-three percent (460/494) of CP donors at the time of plasma donation had a neutralizing IgG titer ≥1:80. Plasmapheresis-related adverse reactions occurred in 2.6% (13/504) of cases; all the reactions were mild and none required therapeutic intervention. Donors' age and COVID-19 severity were positively associated with greater antibody responses. CONCLUSION: This study demonstrates the feasibility and safety of a pilot CP program conducted in Italy. The identification of factors (ie, age and severity of COVID-19) positively associated with higher neutralizing antibody titers at the time of donation may help to optimize the selection of CP donors.
Subject(s)
Blood Donors/statistics & numerical data , COVID-19/therapy , Donor Selection/statistics & numerical data , Adolescent , Adult , Aged , COVID-19/immunology , Donor Selection/methods , Feasibility Studies , Female , Humans , Immunization, Passive , Italy , Male , Middle Aged , Pilot Projects , Plasmapheresis/adverse effects , Plasmapheresis/statistics & numerical data , Retrospective Studies , Young Adult , COVID-19 SerotherapyABSTRACT
BACKGROUND: Convalescent plasma (CP) and hyperimmune plasma (HP) are passive immunotherapies consisting in the infusion of plasma from recovered people into infected patients. Following pre-existing evidence in many other viral diseases, such as SARS, MERS and Ebola, CP and HP have also been proposed for the treatment of COVID-19. Nevertheless, due to the lack of large, well-designed, clinical trials, no clear-cut guidelines exist about what subtype of patient CP and HP should be administered to. CASE PRESENTATION: We have reported the cases of 3 patients, all immunosuppressed and affected by non-severe, prolonged COVID-19. They were treated with HP, whose neutralizing titer was higher than 1/80. The first patient was a 55-year-old male, who had undergone lung transplant. He was under therapy with Tacrolimus and developed non-neutralizing antibodies against SARS-CoV2. The second patient was a 77-year-old female, affected by follicular lymphoma. She had tested positive for SARS-CoV2 after 6 months. The third was a 60-year-old patient, affected by chronic leukemia. He did not develop antibodies after 2-month disease. All 3 patients received HP and had tested negative for SARS-CoV2 within 2 weeks. CONCLUSION: Despite encouraging initial data, no strong evidence exist in support of CP and HP to treat COVID-19. In our experience, although limited due to the reduced number of patients, we found a good safety and efficacy of HP in 3 immuno-deficient subjects. Further data are needed in order to assess whether this subtype of patients may particularly benefit from passive immunization.
Subject(s)
COVID-19/therapy , SARS-CoV-2 , Adult , Aged , Antibodies, Viral , Female , Humans , Immunization, Passive , Immunocompromised Host , Male , Middle Aged , Plasma , RNA, Viral , Treatment Outcome , COVID-19 SerotherapyABSTRACT
In the coronavirus (CoV) disease 2019 (COVID-19) pandemic, highly selective serological testing is essential to define exposure to severe acute respiratory syndrome CoV 2 (SARS-CoV-2). Many tests have been developed, yet with variable speeds to first results, and are of unknown quality, particularly when considering the prediction of neutralizing capacity. The LIAISON SARS-CoV-2 S1/S2 IgG assay was designed to measure antibodies against the SARS-CoV-2 native S1/S2 proteins in a standardized automated chemiluminescence assay. The clinical and analytical performances of the test were validated in an observational study using residual samples (>1,500) with a positive or negative COVID-19 diagnosis. The LIAISON SARS-CoV-2 S1/S2 IgG assay proved to be highly selective and specific and offered semiquantitative measures of serum or plasma levels of anti-S1/S2 IgG with neutralizing activity. The assay's diagnostic sensitivities were 91.3% and 95.7% at >5 or ≥15 days from diagnosis, respectively, and 100% when assessed against a neutralizing assay. The assay's specificity ranged between 97% and 98.5%. The average imprecision of the assay was a <5% coefficient of variation. Assay performance at 2 different cutoffs was evaluated to optimize predictive values. The automated LIAISON SARS-CoV-2 S1/S2 IgG assay brings efficient, sensitive, specific, and precise serological testing to the laboratory, with the capacity to test large amounts of samples per day; first results are available within 35 min, with a throughput of 170 tests/hour. The semiquantitative results provided by the test also associate with the presence of neutralizing antibodies and may provide a useful tool for the large-scale screening of convalescent-phase plasma for safe therapeutic use.
Subject(s)
Antibodies, Viral/blood , Betacoronavirus/immunology , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Serologic Tests , Antibodies, Neutralizing/blood , Automation, Laboratory , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques/methods , Clinical Laboratory Techniques/standards , Clinical Laboratory Techniques/statistics & numerical data , Coronavirus Infections/immunology , Humans , Immunoglobulin G/blood , Pandemics , Pneumonia, Viral/immunology , Reproducibility of Results , SARS-CoV-2 , Sensitivity and Specificity , Serologic Tests/methods , Serologic Tests/standards , Serologic Tests/statistics & numerical data , Spike Glycoprotein, Coronavirus/immunologyABSTRACT
Hyperimmune plasma from Covid-19 convalescent is a potential treatment for severe Covid-19. We conducted a multicenter one arm proof of concept interventional study. Patients with Covid-19 disease with moderate-to-severe Acute Respiratory Distress Syndrome, elevated C-reactive Protein and need for mechanical ventilation and/or CPAP were enrolled. One to three 250-300 ml unit of hyperimmune plasma (neutralizing antibodies titer ≥1:160) were administered. Primary outcome was 7-days hospital mortality. Secondary outcomes were PaO2/FiO2, laboratory and radiologic changes, as well as weaning from mechanical ventilation and safety. The study observed 46 patients from March, 25 to April, 21 2020. Patients were aged 63, 61% male, of them, 30 were on CPAP and 7 intubated. PaO2/FiO2 was 128 (SD 47). Bilateral infiltrates on chest X-ray was present in 36 patients (84%). Symptoms and ARDS duration were 14 (SD 7) and 6 days (SD 3). Three patients (6.5%) died within 7 days as compared to an expected 15% from the National Statistics and 30% from a small concurrent cohort of 23 patients. The upper one-sided 90%CI was 13.9%, allowing to reject the null hypothesis of a 15% mortality. PaO2/FiO2 increased by 112 units (95%CI 82 to142) in survivors, the chest radiogram severity decreased in 23% (95%CI 5% to 42%); CRP, Ferritin and LDH decreased by 60, 36 and 20% respectively. Weaning from CPAP was obtained in 26/30 patients and 3/7 were extubated. Five serious adverse events occurred in 4 patients (2 likely, 2 possible treatment related). In conclusion, Hyperimmune plasma in Covid-19 shows promising benefits, to be confirmed in a randomized controlled trial. This proof of concept study could open to future developments including hyperimmune plasma banking, development of standardized pharmaceutical products and monoclonal antibodies.
Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/mortality , COVID-19/therapy , Hospital Mortality/trends , Immunization, Passive/methods , Aged , Aged, 80 and over , COVID-19/immunology , COVID-19/virology , Female , Humans , Male , Middle Aged , Prognosis , SARS-CoV-2/immunology , Severity of Illness Index , Survival Rate , COVID-19 SerotherapyABSTRACT
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly reached pandemic proportions. Given that the main target of SARS-CoV-2 are lungs leading to severe pneumonia with hyperactivation of the inflammatory cascade, we conducted a prospective study to assess alveolar inflammatory status in patients with moderate to severe COVID-19. METHODS: Diagnostic bronchoalveolar lavage (BAL) was performed in 33 adult patients with SARS-CoV-2 infection by real-time PCR on nasopharyngeal swab admitted to the Intensive care unit (ICU) (n = 28) and to the Intermediate Medicine Ward (IMW) (n = 5). We analyze the differential cell count, ultrastructure of cells and Interleukin (IL)6, 8 and 10 levels. RESULTS: ICU patients showed a marked increase in neutrophils (1.24 × 105 ml- 1, 0.85-2.07), lower lymphocyte (0.97 × 105 ml- 1, 0.024-0.34) and macrophages fractions (0.43 × 105 ml- 1, 0.34-1.62) compared to IMW patients (0.095 × 105 ml- 1, 0.05-0.73; 0.47 × 105 ml- 1, 0.28-1.01 and 2.14 × 105 ml- 1, 1.17-3.01, respectively) (p < 0.01). Study of ICU patients BAL by electron transmission microscopy showed viral particles inside mononuclear cells confirmed by immunostaining with anti-viral capsid and spike antibodies. IL6 and IL8 were significantly higher in ICU patients than in IMW (IL6 p < 0.01, IL8 p < 0.0001), and also in patients who did not survive (IL6 p < 0.05, IL8 p = 0.05 vs. survivors). IL10 did not show a significant variation between groups. Dividing patients by treatment received, lower BAL concentrations of IL6 were found in patients treated with steroids as compared to those treated with tocilizumab (p < 0.1) or antivirals (p < 0.05). CONCLUSIONS: Alveolitis, associated with COVID-19, is mainly sustained by innate effectors which showed features of extensive activation. The burden of pro-inflammatory cytokines IL6 and IL8 in the broncho-alveolar environment is associated with clinical outcome.
Subject(s)
Bronchoalveolar Lavage Fluid/immunology , Coronavirus Infections/immunology , Inflammation/immunology , Interleukin-6/immunology , Interleukin-8/immunology , Leukocytes/immunology , Lung/immunology , Macrophages, Alveolar/immunology , Pneumonia, Viral/immunology , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/therapeutic use , Adrenal Cortex Hormones/therapeutic use , Aged , Alanine/analogs & derivatives , Alanine/therapeutic use , Antibodies, Monoclonal, Humanized/therapeutic use , Antiviral Agents/therapeutic use , Betacoronavirus , Bronchoalveolar Lavage , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/virology , COVID-19 , Coronavirus Infections/drug therapy , Coronavirus Infections/therapy , Drug Combinations , Female , Humans , Hydroxychloroquine/therapeutic use , Intensive Care Units , Interleukin-10/immunology , Italy , Leukocytes, Mononuclear/virology , Lopinavir/therapeutic use , Lung/cytology , Lung/virology , Lymphocytes/immunology , Male , Microscopy, Electron, Transmission , Middle Aged , Neutrophils/immunology , Pandemics , Pneumonia, Viral/therapy , Prognosis , Prospective Studies , Respiration, Artificial/methods , Ritonavir/therapeutic use , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism , Survival Rate , Virion/metabolism , Virion/ultrastructure , COVID-19 Drug TreatmentABSTRACT
Zika virus (ZIKV) is a mosquito-borne flavivirus that emerged recently as a global health threat, causing a pandemic in the Americas. ZIKV infection mostly causes mild disease, but is linked to devastating congenital birth defects and Guillain-Barré syndrome in adults. The high level of cross-reactivity among flaviviruses and their cocirculation has complicated serological approaches to differentially detect ZIKV and dengue virus (DENV) infections, accentuating the urgent need for a specific and sensitive serological test. We previously generated a ZIKV nonstructural protein 1 (NS1)-specific human monoclonal antibody, which we used to develop an NS1-based competition ELISA. Well-characterized samples from RT-PCR-confirmed patients with Zika and individuals exposed to other flavivirus infections or vaccination were used in a comprehensive analysis to determine the sensitivity and specificity of the NS1 blockade-of-binding (BOB) assay, which was established in laboratories in five countries (Nicaragua, Brazil, Italy, United Kingdom, and Switzerland). Of 158 sera/plasma from RT-PCR-confirmed ZIKV infections, 145 (91.8%) yielded greater than 50% inhibition. Of 171 patients with primary or secondary DENV infections, 152 (88.9%) scored negative. When the control group was extended to patients infected by other flaviviruses, other viruses, or healthy donors (n = 540), the specificity was 95.9%. We also analyzed longitudinal samples from DENV-immune and DENV-naive ZIKV infections and found inhibition was achieved within 10 d postonset of illness and maintained over time. Thus, the Zika NS1 BOB assay is sensitive, specific, robust, simple, low-cost, and accessible, and can detect recent and past ZIKV infections for surveillance, seroprevalence studies, and intervention trials.
Subject(s)
Antibodies, Viral/immunology , Enzyme-Linked Immunosorbent Assay/methods , Flavivirus Infections/diagnosis , Viral Nonstructural Proteins/immunology , Zika Virus Infection/diagnosis , Zika Virus/immunology , Adolescent , Antibodies, Blocking/immunology , Antibodies, Monoclonal/immunology , Child , Child, Preschool , Cross Reactions/immunology , Dengue/diagnosis , Dengue/virology , Diagnosis, Differential , Flavivirus Infections/virology , Humans , Prospective Studies , Sensitivity and Specificity , Zika Virus Infection/virologyABSTRACT
We evaluated SARS-CoV-2 RNA and neutralising antibodies in blood donors (BD) residing in the Lodi Red Zone, Italy. Of 390 BDs recruited after 20 February 2020 - when the first COVID-19 case in Lombardy was identified, 91 (23%) aged 19-70 years were antibody positive. Viral RNA was detected in an additional 17 (4.3%) BDs, yielding ca 28% (108/390) with evidence of virus exposure. Five stored samples collected as early as 12 February were seropositive.
Subject(s)
Antibodies, Neutralizing/immunology , Betacoronavirus/immunology , Coronavirus Infections/epidemiology , Pneumonia, Viral/epidemiology , Adult , Aged , Blood Donors , COVID-19 , COVID-19 Testing , COVID-19 Vaccines , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Coronavirus Infections/immunology , Coronavirus Infections/therapy , Female , Humans , Immunization, Passive , Italy/epidemiology , Male , Middle Aged , Neutralization Tests , Pandemics , Pneumonia, Viral/immunology , Pneumonia, Viral/therapy , Prevalence , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2 , Seroepidemiologic Studies , Young Adult , COVID-19 SerotherapyABSTRACT
Broadly neutralizing antibodies reactive against most and even all variants of the same viral species have been described for influenza and HIV-1 (ref. 1). However, whether a neutralizing antibody could have the breadth of range to target different viral species was unknown. Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are common pathogens that cause severe disease in premature newborns, hospitalized children and immune-compromised patients, and play a role in asthma exacerbations. Although antisera generated against either HRSV or HMPV are not cross-neutralizing, we speculated that, because of the repeated exposure to these viruses, cross-neutralizing antibodies may be selected in some individuals. Here we describe a human monoclonal antibody (MPE8) that potently cross-neutralizes HRSV and HMPV as well as two animal paramyxoviruses: bovine RSV (BRSV) and pneumonia virus of mice (PVM). In its germline configuration, MPE8 is HRSV-specific and its breadth is achieved by somatic mutations in the light chain variable region. MPE8 did not result in the selection of viral escape mutants that evaded antibody targeting and showed potent prophylactic efficacy in animal models of HRSV and HMPV infection, as well as prophylactic and therapeutic efficacy in the more relevant model of lethal PVM infection. The core epitope of MPE8 was mapped on two highly conserved anti-parallel ß-strands on the pre-fusion viral F protein, which are rearranged in the post-fusion F protein conformation. Twenty-six out of the thirty HRSV-specific neutralizing antibodies isolated were also found to be specific for the pre-fusion F protein. Taken together, these results indicate that MPE8 might be used for the prophylaxis and therapy of severe HRSV and HMPV infections and identify the pre-fusion F protein as a candidate HRSV vaccine.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Cross Reactions/immunology , Paramyxoviridae Infections/immunology , Paramyxoviridae Infections/virology , Paramyxoviridae/classification , Paramyxoviridae/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/isolation & purification , Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/isolation & purification , Antibodies, Neutralizing/therapeutic use , Antibody Specificity/immunology , Cattle , Epitopes/immunology , Humans , Immunoglobulin Light Chains/chemistry , Immunoglobulin Light Chains/immunology , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/immunology , Metapneumovirus/immunology , Mice , Models, Molecular , Molecular Sequence Data , Murine pneumonia virus/immunology , Paramyxoviridae Infections/prevention & control , Paramyxoviridae Infections/therapy , Pneumovirus Infections/immunology , Pneumovirus Infections/prevention & control , Pneumovirus Infections/virology , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus Infections/prevention & control , Respiratory Syncytial Virus Infections/therapy , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Bovine/immunology , Respiratory Syncytial Virus, Human/immunology , Viral Fusion Proteins/chemistry , Viral Fusion Proteins/immunology , Viral Vaccines/chemistry , Viral Vaccines/immunologyABSTRACT
Blood donation screening for West Nile virus (WNV) was mandatory in the Lazio region in 2017 and 2018 (June-November) according to the national surveillance plan. In these years, all five donations reactive in WNV nucleic acid amplification tests harboured instead Usutu virus (USUV). Clade 'Europe 2' was identified in four blood donations and a 2018 mosquito pool. The cocirculation of WNV and USUV in Lazio warrants increased laboratory support and awareness of possible virus misidentification.
Subject(s)
Blood Donors/statistics & numerical data , Disease Outbreaks/prevention & control , Flavivirus Infections/epidemiology , Flavivirus/isolation & purification , West Nile virus/isolation & purification , Adult , Aged , Animals , Culicidae/virology , Flavivirus/genetics , Flavivirus Infections/diagnosis , Flavivirus Infections/virology , Humans , Italy/epidemiology , Male , Middle Aged , Nucleic Acid Amplification Techniques , Phylogeny , Polymerase Chain Reaction , Sentinel Surveillance , Sequence Analysis , West Nile virus/geneticsABSTRACT
Background: To improve our understanding of the natural history of Zika virus (ZIKV) infection in humans, we described the dynamics of ZIKV RNA shedding in different body fluids and antibody responses in patients with acute infection. Methods: Twenty-nine adults with travel-associated infection and 1 case of sexual transmission were enrolled and followed up with weekly ZIKV RNA testing in blood, urine, saliva, and semen samples and antibody testing. Results: ZIKV RNA was detected in plasma, urine, and saliva of 57%, 93.1%, and 69.2% of participants, with estimated median times to clearance of 11.5 days (interquartile range [IQR] 6-24 days), 24 days (IQR, 17-34), and 14 days (IQR, 8-31), respectively. In 2 pregnant women, ZIKV RNA persisted in blood until delivery of apparently healthy infants. ZIKV RNA was detected in semen of 5 of 10 tested men; median time to clearance was 25 days (IQR 14-29), and the longest time of shedding in semen was 370 days. In flavivirus-naive patients, the median times to detection of ZIKV nonstructural protein 1 (NS1)-specific immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies were estimated as 8 days (IQR, 5-15 days) and 17 days (IQR, 12-26 days), respectively. ZIKV NS1 IgM antibodies were undetectable in patients with previous dengue. Conclusions: Prolonged viremia and ZIKV RNA shedding in urine, saliva, and semen occur frequently in patients with acute ZIKV infection. At the time of diagnosis, about half of patients are ZIKV IgM negative. ZIKV NS1 IgM antibodies remain undetectable in patients with previous dengue. Estimates of the times to viral clearance and seroconversion are useful to optimize diagnostic algorithms.