A comparative seroepidemiologic study of the neutralizing antibody against the virulent standard strains and the Sabin vaccine strains of poliovirus among healthy Japanese

The neutralizing antibody of 160 serum specimens collected in 1978 from healthy residents in five prefectures in Japan was titrated against both the virulent standard strains and the Sabin vaccine strains of three types of poliovirus. Antibody-positive rates with both strains of respective types at a level of 1:4 were comparable in all three types of poliovirus. However, the geometric mean titers (GMTs) obtained against both strains showed statistically significant difference depending on the age-cohort's previous history of exposure to the wild or the vaccine strains of polioviruses: the younger age cohorts showed higher GMTs to the Sabin strains, while adults responded higher to the virulent standard strains. The difference was most pronounced in type 1.     

A novel bivalent Pasteurellosis-RHD vaccine candidate adjuvanted with Montanide ISA70 protects rabbits from lethal challenge

In the present study, a bivalent vaccine against Pasteurella multocida and rabbit hemorrhagic disease virus (RHDV) was formulated with Montanide™ ISA70 oil adjuvant (Seppic, Paris, France). Its efficacy was evaluated and compared to similar monovalent preparations and commercially available monovalent vaccines. White new Zeeland rabbit groups (n = 10) received 2 successive doses of the tested vaccines and were challenged 2 weeks after 2nd dose with Pasteurella multocida and RHDV or either pathogens according to their vaccination schedule. Challenged not-vaccinated group of rabbits (n = 10) was included as a control. The bivalent and monovalent ISA70 preparations were found stable, safe, sterile, pure and of low viscosity. Group 3 (GP3) which received bivalent vaccine showed the highest antibody geometric mean titers against Pasteurella multocida and RHDV evaluated by ELISA and hemagglutination inhibition (HI) respectively. Following virulent challenge; Gp3 rabbits were 90% protected from challenge over other groups that showed 80% protection. Detection of either pathogen in the livers of dead and euthanized rabbits had failed except for non-vaccinated controls. The bivalent vaccine candidate was fully protective. Immunization against both pathogens can be achieved by single vaccination.     

Cold-Adapted Viral Attenuation (CAVA): Highly Temperature Sensitive Polioviruses as Novel Vaccine Strains for a Next Generation Inactivated Poliovirus Vaccine

The poliovirus vaccine field is moving towards novel vaccination strategies. Withdrawal of the Oral Poliovirus Vaccine and implementation of the conventional Inactivated Poliovirus Vaccine (cIPV) is imminent. Moreover, replacement of the virulent poliovirus strains currently used for cIPV with attenuated strains is preferred. We generated Cold-Adapted Viral Attenuation (CAVA) poliovirus strains by serial passage at low temperature and subsequent genetic engineering, which contain the capsid sequences of cIPV strains combined with a set of mutations identified during cold-adaptation. These viruses displayed a highly temperature sensitive phenotype with no signs of productive infection at 37°C as visualized by electron microscopy. Furthermore, decreases in infectious titers, viral RNA, and protein levels were measured during infection at 37°C, suggesting a block in the viral replication cycle at RNA replication, protein translation, or earlier. However, at 30°C, they could be propagated to high titers (9.4–9.9 Log₁₀TCID₅₀/ml) on the PER.C6 cell culture platform. We identified 14 mutations in the IRES and non-structural regions, which in combination induced the temperature sensitive phenotype, also when transferred to the genomes of other wild-type and attenuated polioviruses. The temperature sensitivity translated to complete absence of neurovirulence in CD155 transgenic mice. Attenuation was also confirmed after extended in vitro passage at small scale using conditions (MOI, cell density, temperature) anticipated for vaccine production. The inability of CAVA strains to replicate at 37°C makes reversion to a neurovirulent phenotype in vivo highly unlikely, therefore, these strains can be considered safe for the manufacture of IPV. The CAVA strains were immunogenic in the Wistar rat potency model for cIPV, inducing high neutralizing antibody titers in a dose-dependent manner in response to D-antigen doses used for cIPV. In combination with the highly productive PER.C6 cell culture platform, the stably attenuated CAVA strains may serve as an attractive low-cost and (bio)safe option for the production of a novel next generation IPV.     

Recombinant, live-attenuated tetravalent dengue virus vaccine formulations induce a balanced, broad, and protective neutralizing antibody response against each of the four serotypes in rhesus monkeys

Three tetravalent vaccine (TV) formulations of previously described monovalent dengue (DEN) virus vaccine candidates were compared to a tetravalent formulation of wild-type DEN viruses (T-wt) for replication in SCID mice transplanted with human liver cells (SCID-HuH-7) or for replication and immunogenicity in rhesus monkeys. TV-1 consists of recombinant DEN1, -2, -3, and -4, each with a 30-nucleotide deletion in the 3' untranslated region (Delta30). TV-2 consists of rDEN1Delta30, rDEN4Delta30, and two antigenic chimeric viruses, rDEN2/4Delta30 and rDEN3/4Delta30, both also bearing the Delta30 mutation. TV-3 consists of rDEN1Delta30, rDEN2Delta30, rDEN4Delta30, and a 10-fold higher dose of rDEN3/4Delta30. TV-1 and TV-2 were attenuated in SCID-HuH-7 mice with minimal interference in replication among the virus components. TV-1, -2, and -3 were attenuated in rhesus monkeys as measured by duration and peak of viremia. Each monkey immunized with TV-1 and TV-3 seroconverted to the four DEN components by day 28 with neutralization titers ranging from 1:52 to 1:273 and 1:59 to 1:144 for TV-1 and TV-3, respectively. TV-2 induced low antibody titers to DEN2 and DEN3, but a booster immunization after 4 months increased the neutralizing antibody titers to greater than 1:100 against each serotype and elicited broad neutralizing activity against 19 of 20 DEN subtypes. A single dose of TV-2 induced protection against wild-type DEN1, DEN3, and DEN4 challenge, but not DEN2. However, two doses of TV-2 or TV-3 induced protection against DEN2 challenge. Two tetravalent formulations, TV-2 and TV-3, possess properties of a successful DEN vaccine and can be considered for evaluation in clinical trials.     

Protection of vaccinia-primed macaques against SIVmne infection by combination immunization with recombinant vaccinia virus and SIVmne gp160

Two Macaca fascicularis with preexisting immunity to vaccinia virus were immunized twice with recombinant vaccinia virus expressing SIV_mne gp160. Their SIV-specific antibody responses were lower than that of vaccinia-naive animals immunized similarly. Upon repeated boosting with gp160, the SIV-specific antibody titers in vaccinia-primed animals reached similar levels as vaccinia-naive animals and with comparable neutralizing titers. Both animals were protected against repeated intravenous challenge with low-dose SIV_mne E11S. These results are significant because SIV_mne E11S infection in M. fascicularis is pathogenic and leads to AIDS-like diseases.     

Evaluation in mice of the immunogenicity and protective efficacy of a tetravalent subunit vaccine candidate against dengue virus

A dengue vaccine must induce protective immunity against the four serotypes of the virus. Our group has developed chimeric proteins consisting of the protein P64k from Neisseria meningitidis and the domain III from the four viral envelope proteins. In this study, the immunogenicity of a tetravalent vaccine formulation using aluminum hydroxide as adjuvant was evaluated in mice. After three doses, neutralizing antibody titers were detected against the four viral serotypes, the lowest seroconversion rate being against dengue virus serotype 4. One month after the last dose, immunized animals were challenged with infective virus, and partial but statistically significant protection was found to have been achieved. Based on these results, further studies in mice and non‐human primates using this tetravalent formulation in a prime‐boost strategy with attenuated viruses are strongly recommended.     

Clinicopathological and immunological studies on toxoids vaccine as a successful alternative in controlling clostridial infection in broilers

The present study was conducted to evaluate the efficacy and safety of three vaccination regimes of Clostridium perfringens (C. perfringens) type A, C and combined A&C toxoids based on their clinical signs and immunological effects. The vaccines were administered two times at two weeks interval (7 & 21 days old), then the birds were challenged (35 days old) with virulent strains of C. perfringens type A, C and combined A&C. Blood samples were taken one week after the first and second vaccination as well as after challenge. The evaluated parameters in this study included: clinical signs, gross intestinal lesions, complete blood count (CBC), serum protein, liver profiles, and enzyme-linked immunosorbent assay (ELISA) test for detecting serum antibody titers. The results revealed that immunization of broilers with C. perfringens type A, C and combined A&C toxoids resulted in a significant decrease in numbers of chickens with intestinal lesions particularly with the A&C toxoids vaccine. Results of the CBC values were significantly increased in all treated groups and challenged groups. Total leukocytic count decreased in challenged non vaccinated group while increased in challenged vaccinated birds. Results of biochemical assays implicated that there were a significant increase in serum protein and liver profiles. ELISA results explored a significant increase in antibody titers after immunization of broilers with C. perfringens type A, C and combined A&C toxoids particularly after the second dose of vaccination. We concluded that immunization of broilers with toxoid vaccines particularly the combined type A & C is safe, well-tolerated and can protect broiler chickens against necrotic enteritis particularly after the second booster dose of the vaccine.     

The Immunodominance Change and Protection of CD4+ T-Cell Responses Elicited by an Envelope Protein Domain III-Based Tetravalent Dengue Vaccine in Mice

Dengue is the leading cause of mosquito-borne viral infections and no vaccine is available now. Envelope protein domain III (ED3) is the major target for the binding of dengue virus neutralizing antibodies; however, the ED3-specifc T-cell response is less well understood. To investigate the T-cell responses to four serotypes of dengue virus (DENV-1 to 4), we immunized mice using either a tetravalent ED3-based DNA or protein vaccine, or combined both as a DNA prime-protein boost strategy (prime-boost). A significant serotype-dependent IFN-γ or IL-4 response was observed in mice immunized with either the DNA or protein vaccine. The IFN-γ response was dominant to DENV-1 to 3, whereas the IL-4 response was dominant to DENV-4. Although the similar IgG titers for the four serotypes were observed in mice immunized with the tetravalent vaccines, the neutralizing antibody titers varied and followed the order of 2 = 3>1>4. Interestingly, the lower IFN-γ response to DENV-4 is attributable to the immunodominance change between two CD4⁺ T-cell epitopes; one T-cell epitope located at E_349-363 of DENV-1 to 3 was more immunogenic than the DENV-4 epitope E_313-327. Despite DENV-4 specific IFN-γ responses were suppressed by immunodominance change, either DENV-4-specific IFN-γ or neutralizing antibody responses were still recalled after DENV-4 challenge and contributed to virus clearance. Immunization with the prime-boost elicited both IFN-γ and neutralizing antibody responses and provided better protection than either DNA or protein immunization. Our findings shed light on how ED3-based tetravalent dengue vaccines sharpen host CD4 T-cell responses and contribute to protection against dengue virus.     

Defining levels of dengue virus serotype-specific neutralizing antibodies induced by a live attenuated tetravalent dengue vaccine (TAK-003)

The four dengue virus serotypes (DENV1-4) infect several hundred million people each year living in tropical and sub-tropical regions. Clinical development of DENV vaccines is difficult because immunity to a single serotype increases risk of severe disease during a second infection with a new serotype. Leading vaccines are based on tetravalent formulations to induce simultaneous and balanced protective immunity to all 4 serotypes. TAK-003 is a tetravalent live attenuated dengue vaccine candidate developed by Takeda Vaccines Inc, which is currently being evaluated in phase 3 efficacy trials. Here, we use antibody depletion methods and chimeric, epitope transplant DENVs to characterize the specificity of neutralizing antibodies in dengue-naïve adults and non-human primates immunized with TAK-003. Our results demonstrate that TAK-003 induced high levels of DENV2 neutralizing antibodies that recognized unique (type-specific) epitopes on DENV2. In contrast, most vaccinated subjects developed lower levels of DENV1, DENV3 and DENV4 neutralizing antibodies that mainly targeted epitopes that were conserved (cross-reactive) between serotypes.Trial Registration: ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02425098","term_id":"NCT02425098"}}NCT02425098.     

Detection of mutants in polio vaccine viruses using pooled antipoliovirus monoclonal antibodies.

We prepared six monoclonal antibodies (mAbs) for type 1 polioviruses, and analysed their neutralizing specificities for use in safety tests in oral poliomyelitis vaccine (OPV) production. Pools of two or more individual mAbs showed high neutralizing activity against high-titre (approximately 10(7) CCID (50)/25 microl) of Sabin type 1 virus. It was demonstrated that the pooled mAbs can be utilized effectively in detection tests of adventitious viruses, which are among the safety tests in OPV production. Moreover, some pooled mAbs were shown to be capable of detecting very small amounts of type 1 virulent viruses and mutants in high-titre Sabin type 1 virus suspensions. Neutralizing antibody titres of these pooled mAbs decreased with increasing numbers of mutants containing neurovirulent activity in high-titre Sabin type 1 viruses which were repeatedly passaged in culture. It is expected that these pooled mAbs will contribute greatly to safety tests for OPV production.     

Estimation of post-vaccination antibody titre against goat pox and determination of protective antibody titre

Goats maintained in farm/under rural condition by individual owners with definite history of vaccination were vaccinated with Freeze Dried Tissue Culture Goat Pox Vaccine and antibody titre was determined up to 1 year post-vaccination period. A few experimental goats were vaccinated and subsequently challenged with isolated virulent field virus and antibody titre of vaccinated animals observed at different intervals was compared with the antibody titre observed in the experimental goats. Post-vaccination serum neutralizing antibody titre was 1:32 at 1 year post-vaccination. All the vaccinated experimental goats withstood virulent field virus challenge on 21st day with serum neutralizing antibody titre of 1:16.     

Dengue 2 PDK-53 virus as a chimeric carrier for tetravalent dengue vaccine development

Attenuation markers of the candidate dengue 2 (D2) PDK-53 vaccine virus are encoded by mutations that reside outside of the structural gene region of the genome. We engineered nine dengue virus chimeras containing the premembrane (prM) and envelope (E) genes of wild-type D1 16007, D3 16562, or D4 1036 virus within the genetic backgrounds of wild-type D2 16681 virus and the two genetic variants (PDK53-E and PDK53-V) of the D2 PDK-53 vaccine virus. Expression of the heterologous prM-E genes in the genetic backgrounds of the two D2 PDK-53 variants, but not that of wild-type D2 16681 virus, resulted in chimeric viruses that retained PDK-53 characteristic phenotypic markers of attenuation, including small plaque size and temperature sensitivity in LLC-MK(2) cells, limited replication in C6/36 cells, and lack of neurovirulence in newborn ICR mice. Chimeric D2/1, D2/3, and D2/4 viruses replicated efficiently in Vero cells and were immunogenic in AG129 mice. Chimeric D2/1 viruses protected adult AG129 mice against lethal D1 virus challenge. Two tetravalent virus formulations, comprised of either PDK53-E- or PDK53-V-vectored viruses, elicited neutralizing antibody titers in mice against all four dengue serotypes. These antibody titers were similar to the titers elicited by monovalent immunizations, suggesting that viral interference did not occur in recipients of the tetravalent formulations. The results of this study demonstrate that the unique attenuation loci of D2 PDK-53 virus make it an attractive vector for the development of live attenuated flavivirus vaccines.     

Exploring of Primate Models of Tick-Borne Flaviviruses Infection for Evaluation of Vaccines and Drugs Efficacy

Tick-borne encephalitis virus (TBEV) is one of the most prevalent and medically important tick-borne arboviruses in Eurasia. There are overlapping foci of two flaviviruses: TBEV and Omsk hemorrhagic fever virus (OHFV) in Russia. Inactivated vaccines exist only against TBE. There are no antiviral drugs for treatment of both diseases. Optimal animal models are necessary to study efficacy of novel vaccines and treatment preparations against TBE and relative flaviviruses. The models for TBE and OHF using subcutaneous inoculation were tested in Cercopithecus aethiops and Macaca fascicularis monkeys with or without prior immunization with inactivated TBE vaccine. No visible clinical signs or severe pathomorphological lesions were observed in any monkey infected with TBEV or OHFV. C. aethiops challenged with OHFV showed massive hemolytic syndrome and thrombocytopenia. Infectious virus or viral RNA was revealed in visceral organs and CNS of C. aethiops infected with both viruses; however, viremia was low. Inactivated TBE vaccines induced high antibody titers against both viruses and expressed booster after challenge. The protective efficacy against TBE was shown by the absence of virus in spleen, lymph nodes and CNS of immunized animals after challenge. Despite the absence of expressed hemolytic syndrome in immunized C. aethiops TBE vaccine did not prevent the reproduction of OHFV in CNS and visceral organs. Subcutaneous inoculation of M. fascicularis with two TBEV strains led to a febrile disease with well expressed viremia, fever, and virus reproduction in spleen, lymph nodes and CNS. The optimal terms for estimation of the viral titers in CNS were defined as 8–16 days post infection. We characterized two animal models similar to humans in their susceptibility to tick-borne flaviviruses and found the most optimal scheme for evaluation of efficacy of preventive and therapeutic preparations. We also identified M. fascicularis to be more susceptible to TBEV than C. aethiops.     

AIDS vaccination studies using an ex vivo feline immunodeficiency virus model: reevaluation of neutralizing antibody levels elicited by a protective and a nonprotective vaccine after removal of antisubstrate cell antibodies

In the feline immunodeficiency virus system, immunization with a fixed-infected-cell vaccine conferred protection against virulent homologous challenge but the immune effectors involved remained elusive. In particular, few or no neutralizing antibodies were detected in sera from vaccinated cats. Here we show that, when preadsorbed with selected feline cells, the same sera revealed clearly evident virus-neutralizing activity. Because high titers of neutralizing antibody in cell-adsorbed sera from 23 cats immunized with fixed-infected-cell or whole-inactivated-virus vaccines correlated with protection, it is likely that they were more important for protection than formerly realized. In vitro, the fixed-cell vaccine efficiently removed neutralizing antibody from immune sera while the whole-inactivated-virus vaccine was much less effective.     

Vaccination with SARS-CoV-2 variants of concern protects mice from challenge with wild-type virus

Vaccines against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have been highly efficient in protecting against Coronavirus Disease 2019 (COVID-19). However, the emergence of viral variants that are more transmissible and, in some cases, escape from neutralizing antibody responses has raised concerns. Here, we evaluated recombinant protein spike antigens derived from wild-type SARS-CoV-2 and from variants B.1.1.7, B.1.351, and P.1 for their immunogenicity and protective effect in vivo against challenge with wild-type SARS-CoV-2 in the mouse model. All proteins induced high neutralizing antibodies against the respective viruses but also induced high cross-neutralizing antibody responses. The decline in neutralizing titers between variants was moderate, with B.1.1.7-vaccinated animals having a maximum fold reduction of 4.8 against B.1.351 virus. P.1 induced the most cross-reactive antibody responses but was also the least immunogenic in terms of homologous neutralization titers. However, all antigens protected from challenge with wild-type SARS-CoV-2 in a mouse model.

This study explores the immune response induced by wild type and variant SARS-CoV-2 spike proteins, and the protection that these immune responses provide against challenge with wild type virus in the mouse model.     

Characteristics of Memory B Cells Elicited by a Highly Efficacious HPV Vaccine in Subjects with No Pre-existing Immunity

Licensed human papillomavirus (HPV) vaccines provide near complete protection against the types of HPV that most commonly cause anogenital and oropharyngeal cancers (HPV 16 and 18) when administered to individuals naive to these types. These vaccines, like most other prophylactic vaccines, appear to protect by generating antibodies. However, almost nothing is known about the immunological memory that forms following HPV vaccination, which is required for long-term immunity. Here, we have identified and isolated HPV 16-specific memory B cells from female adolescents and young women who received the quadrivalent HPV vaccine in the absence of pre-existing immunity, using fluorescently conjugated HPV 16 pseudoviruses to label antigen receptors on the surface of memory B cells. Antibodies cloned and expressed from these singly sorted HPV 16-pseudovirus labeled memory B cells were predominantly IgG (>IgA>IgM), utilized diverse variable genes, and potently neutralized HPV 16 pseudoviruses in vitro despite possessing only average levels of somatic mutation. These findings suggest that the quadrivalent HPV vaccine provides an excellent model for studying the development of B cell memory; and, in the context of what is known about memory B cells elicited by influenza vaccination/infection, HIV-1 infection, or tetanus toxoid vaccination, indicates that extensive somatic hypermutation is not required to achieve potent vaccine-specific neutralizing antibody responses.     

A duplex vibriocidal assay to simultaneously measure bactericidal antibody titers against Vibrio cholerae O1 Inaba and Ogawa serotypes

Currently available cholera vaccines are formulated with killed-whole cells of Vibrio cholerae O1 Inaba and Ogawa serotypes. A serum vibriocidal assay has been widely used to evaluate the immunogenicity of cholera vaccines in clinical trials. In this study, we developed a duplex vibriocidal assay to obtain vibriocidal antibody titers against both serotypes simultaneously. Initially, serial dilutions of serum from vaccinees were incubated with guinea pig complements along with both streptomycin-resistant Inaba and ampicillin-resistant Ogawa strains for 1 h. The mixture was then inoculated on separate agar plates containing each antibiotic to selectively culture each corresponding serotype and incubated at 37 °C for 16 to 20 h. Bacterial colonies were enumerated using an automated colony counting system to obtain the vibriocidal antibody titers defined by the reciprocal of serum dilution inhibiting bacterial growth by 50%. Performance of the duplex vibriocidal assay was examined by comparison with a single serotype vibriocidal assay using 20 clinical sera consisting of ten-paired sera prepared at pre- and post-vaccination. Both assays showed a good correlation for vibriocidal titers against the two serotypes, respectively, as determined by Pearson correlation coefficient (r) and regression coefficient (β) analyses; r = 0.998, β = 1.003 for Inaba and r = 0.997, β = 0.999 for Ogawa, respectively. The duplex vibriocidal assay can diminish the amount of sera required for the assay and enhance assay efficiency in terms of time, labor intensity, and expenditure.     

A novel dengue vaccine candidate that induces cross-neutralizing antibodies and memory immunity

A novel dengue vaccine candidate comprised of a consensus dengue virus envelope protein domain III (cED III) was developed to fight against dengue virus infection. The amino acid sequence of this novel cED III was obtained by alignment of amino acid sequences from different isolates of the four serotypes of dengue viruses. A proof-of-concept study demonstrated that BALB/c mice immunized with the recombinant cED III developed neutralizing antibodies against all serotypes of dengue virus. Moreover, formulation of recombinant cED III with aluminum phosphate could induce long-lasting antibody responses and anamnestic neutralizing antibody responses following challenge with dengue virus at week 28 after priming. These results demonstrate the possibility of developing a single tetravalent vaccine against dengue viral infections.     

Heterosubtypic antibody response elicited with seasonal influenza vaccine correlates partial protection against highly pathogenic H5N1 virus

Background

The spread of highly pathogenic avian influenza (HPAI) H5N1 virus in human remains a global health concern. Heterosubtypic antibody response between seasonal influenza vaccine and potential pandemic influenza virus has important implications for public health. Previous studies by Corti et al. and by Gioia et al. demonstrate that heterosubtypic neutralizing antibodies against the highly pathogenic H5N1 virus can be elicited with a seasonal influenza vaccine in humans. However, whether such response offers immune protection against highly pathogenic H5N1 virus remained to be determined.

Methodology/Principal Findings

In this study, using a sensitive influenza HA (hemagglutinin) and NA (neuraminidase) pseudotype-based neutralization (PN) assay we first confirmed that low levels of heterosubtypic neutralizing antibody response against H5N1 virus were indeed elicited with seasonal influenza vaccine in humans. We then immunized mice with the seasonal influenza vaccine and challenged them with lethal doses of highly pathogenic H5N1 virus. As controls, we immunized mice with homosubtypic H5N1 virus like particles (VLP) or PBS and challenged them with the same H5N1 virus. Here we show that low levels of heterosubtypic neutralizing antibody response were elicited with seasonal influenza vaccine in mice, which were significantly higher than those in PBS control. Among them 2 out of 27 whose immune sera exhibited similar levels of neutralizing antibody response as VLP controls actually survived from highly pathogenic H5N1 virus challenge.

Conclusions/Significance

Therefore, we conclude that low levels of heterosubtypic neutralizing antibody response are indeed elicited with seasonal influenza vaccine in humans and mice and at certain levels such response offers immune protection against severity of H5N1 virus infection.     

Time dependent decline of neutralizing antibody titers in COVID-19 patients from Pune,India and evidence of reinfection

PurposeTo assess modulation of neutralizing antibody titers in COVID-19 patients and understand association of variables such as age, presence of comorbidity, BMI and gender with antibody titers.MethodsPatients (n = 100) diagnosed from 20th March 2020 to 17th August 2020 and treated at two large hospitals from Pune, India were included and followed up (clinical and serologic) for varied periods. IgG-anti-SARS-CoV-2 (Spike protein-based ELISA) and neutralizing antibody titers (NAb, PRNT) were determined in all the samples.ResultsOf the 100 patients enrolled initially (median 60 days of diagnosis), follow up samples were collected from 70 patients (median 106 days of diagnosis). Overall, NAb titers reduced significantly (p < 0.001) and as early as 3–4 months. During two visits, 20% and 7.1% patients reported some symptoms. At the first visit, NAb titers were higher in patients with severe disease (p < 0.001), comorbidities (p < 0.005), age <50 years (p < 0.05) and male gender (p < 0.05). Multivariate analysis identified older age (p < 0.001), duration post-diagnosis and female gender as independent variables influencing NAb titers (negative correlation, p < 0.05). During the follow-up, reduction in NAb titers was recorded in patients with comorbidity (p < 0.05), mild disease (p < 0.05), age <50 years (p < 0.05), higher BMI (p < 0.05) and male gender (p < 0.001). Serology identified six cases of asymptomatic reinfections.ConclusionsDecline of NAb titers was associated with age <50 years, mild disease, comorbidities, higher BMI and male gender. At the time of follow up, 8/70 (11.4%) patients lacked neutralizing antibodies. Evidence of 6 probable asymptomatic reinfections suggests waning of immunity, but, probable protection from clinical disease needing hospitalization.