Neutralizing Ab Titers Research Articles

A new mouse model expressing human ACE2, TMPRSS2, and FCGRT for evaluating human antibody responses to SARS-CoV-2

Abstract Protection against viruses is most often measured by levels of plasma binding and neutralizing antibodies (Abs) in vitro, which do not capture the full picture of Ab immunity, including Fc functions. The continuing evolution of SARS-CoV-2 underscores the need to understand protection mediated by Abs. We generated triple knock-in (TKI) mice expressing human ACE2, TMPRSS2, and FCGRT in place of murine genes. A human anti-SARS-CoV-2 IgG1 with a half-life extending mutation but not wild-type IgG1 exhibited long half-life and protected against BA.2 infection in lungs, thereby validating our TKI mice for evaluating human Ab clearance and function. We next evaluated protective efficacy of plasma samples from vaccinated San Diegans against Delta or BA.5 by performing passive transfer experiments. Treatment with pooled plasma from vaccination plus infection but not triple vaccination individuals cleared infectious Delta in lungs of TKI mice, although Delta-binding and -neutralizing Ab titers in vitro were similar between the two groups of people. In contrast, neither group’s plasma impacted lung viral burden upon BA.5 challenge. Thus, Abs elicited by vaccination plus infection but not triple vaccination reduce Delta infection in this mouse model, and Abs from both groups are ineffective against BA.5. These results are consistent with epidemiological data, and show that in vitro Ab titers do not predict in vivo protection. Our TKI mouse model represents an important tool to test efficacy of human monoclonal Abs against SARS-CoV-2/related CoVs, predict human polyclonal Ab-mediated protection against newly emerging variants/CoVs, and dissect mechanisms of Ab-mediated protection in vivo. Results will aid development of pan CoV vaccines.

Evaluation of the immunogenicity and efficacy of an rVSV vaccine against Zika virus infection in macaca nemestrina.

Zika virus (ZIKV) is a mosquito-borne flavivirus that causes an acute febrile illness. ZIKV can be transmitted between sexual partners and from mother to fetus. Infection is strongly associated with neurologic complications in adults, including Guillain-Barré syndrome and myelitis, and congenital ZIKV infection can result in fetal injury and congenital Zika syndrome (CZS). Development of an effective vaccine is imperative to protect against ZIKV vertical transmission and CZS. Recombinant Vesicular Stomatitis virus (rVSV) is a highly effective and safe vector for the delivery of foreign immunogens for vaccine purposes. Here, we evaluate an rVSV vaccine expressing the full length pre-membrane (prM) and ZIKV envelope (E) proteins (VSV-ZprME), shown to be immunogenic in murine models of ZIKV infection, for its capacity to induce immune responses in nonhuman primates. Moreover, we assess the efficacy of the rVSVΔM-ZprME vaccine in the protection of pigtail macaques against ZIKV infection. Administration of the rVSVΔM-ZprME vaccine was safe, but it did not induce robust anti-ZIKV T-cell responses, IgM or IgG antibodies, or neutralizing antibodies in most animals. Post ZIKV challenge, animals that received the rVSVΔM control vaccine lacking ZIKV antigen had higher levels of plasma viremia compared to animals that received the rVSVΔM-ZprME vaccine. Anti-ZIKV neutralizing Ab titers were detected in a single animal that received the rVSVΔM-ZprME vaccine that was associated with reduced plasma viremia. The overall suboptimal ZIKV-specific cellular and humoral responses post-immunization indicates the rVSVΔM-ZprME vaccine did not elicit an immune response in this pilot study. However, recall antibody response to the rVSVΔM-ZprME vaccine indicates it may be immunogenic and further developments to the vaccine construct could enhance its potential as a vaccine candidate in a nonhuman primate pre-clinical model.

The accelerated waning of immunity and reduced effect of booster in patients treated with bDMARD and tsDMARD after SARS-CoV-2 mRNA vaccination.

This study aimed to assess the duration of humoral responses after two doses of SARS-CoV-2 mRNA vaccines in patients with inflammatory joint diseases and IBD and booster vaccination compared with healthy controls. It also aimed to analyze factors influencing the quantity and quality of the immune response. We enrolled 41 patients with rheumatoid arthritis (RA), 35 with seronegative spondyloarthritis (SpA), and 41 suffering from inflammatory bowel disease (IBD), excluding those receiving B-cell-depleting therapies. We assessed total anti-SARS-CoV-2 spike antibodies (Abs) and neutralizing Ab titers 6 months after two and then after three doses of mRNA vaccines compared with healthy controls. We analyzed the influence of therapies on the humoral response. Patients receiving biological or targeted synthetic disease-modifying antirheumatic drugs (b/tsDMARDs) showed reduced anti-SARS-CoV-2 S Abs and neutralizing Ab titers compared with HC or patients receiving conventional synthetic (cs)DMARDs 6 months after the first two vaccination doses. Anti-SARS-CoV-2 S titers of patients with b/tsDMARDs declined more rapidly, leading to a significant reduction in the duration of vaccination-induced immunity after two doses of SARS-CoV-2 mRNA vaccines. While 23% of HC and 19% of patients receiving csDMARDs were without detectable neutralizing Abs 6 months after the first two vaccination doses, this number was 62% in patients receiving b/tsDMARDs and 52% in patients receiving a combination of csDMARDs and b/tsDMARDs. Booster vaccination led to increased anti-SARS-CoV-2 S Abs in all HC and patients. However, anti-SARS-CoV-2 S Abs after booster vaccination was diminished in patients receiving b/tsDMARDs, either alone or in combination with csDMARDs compared to HC. Patients receiving b/tsDMARDs have significantly reduced Abs and neutralizing Ab titers 6 months after mRNA vaccination against SARS-CoV-2. This was due to a faster decline in Ab levels, indicating a significantly reduced duration of vaccination-induced immunity compared with HC or patients receiving csDMARDs. In addition, they display a reduced response to a booster vaccination, warranting earlier booster vaccination strategies in patients under b/tsDMARD therapy, according to their specific Ab levels.

Rotavirus A Inoculation and Oral Vitamin A Supplementation of Vitamin A Deficient Pregnant Sows Enhances Maternal Adaptive Immunity and Passive Protection of Piglets against Virulent Rotavirus A.

The aim of this study was to determine the impact of vitamin A deficiency (VAD)/supplementation (±VA) and group A RV (RVA) maternal immunization of RVA seropositive multiparous pregnant sows, on their immune responses (anamnestic response) and on passive protection of their piglets against RVA challenge. Our results showed that VAD- mock sows had increased RVA RNA shedding at 1-5 days post piglet RVA challenge, and their litters had increased RVA shedding and diarrhea frequency throughout the experiment. VAD decreased memory B cell frequencies while VA supplementation increased RVA specific IgA/IgG antibody (Ab) secreting cell (ASC) numbers in blood, milk, and tissues of RVA inoculated VAD sows. The increased numbers of RVA specific IgA/IgG ASCs in blood, milk/colostrum, intestinal contents, and tissues in VA supplemented VAD sows, suggest a role of VA in B cell immunity and trafficking to tissues. We also observed that RVA inoculated sows had the highest viral neutralizing Ab titers in serum and milk while VA supplementation of VAD sows and RVA inoculation increased IgA+ B cell frequencies in sow colostrum. In summary, we demonstrated that daily oral VA-supplementation (2nd trimester-throughout lactation) to RVA inoculated VAD sows improved the function of their gut-mammary-IgA immunological axis, reducing viral RNA shedding, diarrhea, and increasing weight gain in suckling piglets.

Evidence for the heterologous benefits of prior BCG vaccination on COVISHIELD™ vaccine-induced immune responses in SARS-CoV-2 seronegative young Indian adults.

This proof-of-concept study tested if prior BCG revaccination can qualitatively and quantitively enhance antibody and T-cell responses induced by Oxford/AstraZeneca ChAdOx1nCoV-19 or COVISHIELD™, an efficacious and the most widely distributed vaccine in India. We compared COVISHIELD™ induced longitudinal immune responses in 21 BCG re-vaccinees (BCG-RV) and 13 BCG-non-revaccinees (BCG-NRV), all of whom were BCG vaccinated at birth; latent tuberculosis negative and SARS-CoV-2 seronegative prior to COVISHIELD™ vaccination. Compared to BCG-NRV, BCG-RV displayed significantly higher and persistent spike-specific neutralizing (n) Ab titers and polyfunctional CD4+ and CD8+ T-cells for eight months post COVISHIELD™ booster, including distinct CD4+IFN-γ+ and CD4+IFN-γ- effector memory (EM) subsets co-expressing IL-2, TNF-α and activation induced markers (AIM) CD154/CD137 as well as CD8+IFN-γ+ EM,TEMRA (T cell EM expressing RA) subset combinations co-expressing TNF-α and AIM CD137/CD69. Additionally, elevated nAb and T-cell responses to the Delta mutant in BCG-RV highlighted greater immune response breadth. Mechanistically, these BCG adjuvant effects were associated with elevated markers of trained immunity, including higher IL-1β and TNF-α expression in CD14+HLA-DR+monocytes and changes in chromatin accessibility highlighting BCG-induced epigenetic changes. This study provides first in-depth analysis of both antibody and memory T-cell responses induced by COVISHIELD™ in SARS-CoV-2 seronegative young adults in India with strong evidence of a BCG-induced booster effect and therefore a rational basis to validate BCG, a low-cost and globally available vaccine, as an adjuvant to enhance heterologous adaptive immune responses to current and emerging COVID-19 vaccines.

Anti-SARS-CoV-2 Spike Antibody Titers and Neutralizing Antibodies in Vaccinated Rheumatoid Arthritis Patients.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A serological test is used to assess the efficacy of vaccination. It has been reported that anti-SARS-CoV-2 spike (S) and neutralizing antibody (Ab) levels are lower following vaccination in patients with rheumatic disease. Here, we investigated anti-SARS-CoV-2 S and neutralizing Abs in vaccinated rheumatoid arthritis (RA) patients in Japan. Anti-SARS-CoV-2 S and neutralizing Abs were quantified in 101 RA patients and 117 controls. Anti-SARS-CoV-2 S Ab levels were lower in RA patients than both earlier after vaccination in controls (mean RA 324.1 ± 591.8 SDM vs. control 1216.6 ± 854.4 [U/mL], p < 0.0001) and later after vaccination (324.1 ± 591.8 vs. 582.0 ± 415.6 [U/mL], p = 0.0002). The interval between vaccination of the RA patients and serum collection was longer than for controls early after vaccination (142.1 ± 31.6 vs. 98.3 ± 11.2 [days], p < 0.0001), but shorter than the later sample from the controls (142.1 ± 31.6 vs. 257.3 ± 11.2 [days], p < 0.0001). Importantly, anti-SARS-CoV-2 neutralizing Ab titers in RA patients were higher than in either early or later control samples (10.7 ± 4.9 vs. 8.6 ± 6.6 [%], p = 0.0072, and 10.7 ± 4.9 vs. 3.1 ± 3.7 [%], p < 0.0001, respectively). Anti-SARS-CoV-2 S Ab titers in vaccinated RA patients were lower than in controls, but they were influenced by other clinical manifestations. Anti-SARS-CoV-2 neutralizing Ab levels were independently increased in RA.

Zika virus-like particle vaccine fusion loop mutation increases production yield but fails to protect AG129 mice against Zika virus challenge.

Zika virus (ZIKV) is a mosquito-borne flavivirus with maternal infection associated with preterm birth, congenital malformations, and fetal death, and adult infection associated with Guillain-Barré syndrome. Recent widespread endemic transmission of ZIKV and the potential for future outbreaks necessitate the development of an effective vaccine. We developed a ZIKV vaccine candidate based on virus-like-particles (VLPs) generated following transfection of mammalian HEK293T cells using a plasmid encoding the pre-membrane/membrane (prM/M) and envelope (E) structural protein genes. VLPs were collected from cell culture supernatant and purified by column chromatography with yields of approximately 1-2mg/L. To promote increased particle yields, a single amino acid change of phenylalanine to alanine was made in the E fusion loop at position 108 (F108A) of the lead VLP vaccine candidate. This mutation resulted in a modest 2-fold increase in F108A VLP production with no detectable prM processing by furin to a mature particle, in contrast to the lead candidate (parent). To evaluate immunogenicity and efficacy, AG129 mice were immunized with a dose titration of either the immature F108A or lead VLP (each alum adjuvanted). The resulting VLP-specific binding antibody (Ab) levels were comparable. However, geometric mean neutralizing Ab (nAb) titers using a recombinant ZIKV reporter were significantly lower with F108A immunization compared to lead. After virus challenge, all lead VLP-immunized groups showed a significant 3- to 4-Log10 reduction in mean ZIKV RNAemia levels compared with control mice immunized only with alum, but the RNAemia reduction of 0.5 Log10 for F108A groups was statistically similar to the control. Successful viral control by the lead VLP candidate following challenge supports further vaccine development for this candidate. Notably, nAb titer levels in the lead, but not F108A, VLP-immunized mice inversely correlated with RNAemia. Further evaluation of sera by an in vitro Ab-dependent enhancement assay demonstrated that the F108A VLP-induced immune sera had a significantly higher capacity to promote ZIKV infection in FcγR-expressing cells. These data indicate that a single amino acid change in the fusion loop resulted in increased VLP yields but that the immature F108A particles were significantly diminished in their capacity to induce nAbs and provide protection against ZIKV challenge.

Development of a protective inactivated vaccine against Crimean–Congo hemorrhagic fever infection

Crimean–Congo hemorrhagic fever (CCHF) is an emerging zoonotic infectious disease caused by Crimean–Congo hemorrhagic fever virus (CCHFV). The first clinical CCHF infection was described in 1944 in the Crimean Peninsula, exclusively in humans, with case-fatality rates exceeding 30%. The increasing number of cases, high mortality rate, and lack of effective therapy make CCHF a serious threat to public health and a potential bioterrorism agent. The present study evaluated the development, immunogenicity, and immune response durations for cell-culture-derived inactivated vaccine (CCVax) formulations in comparison with those of mouse-brain-derived vaccine (MBVax) formulations. In this study, the Kelkit06 CCHF virus strain was propagated in both suckling mice and Vero E6 cells, and purified with a sucrose gradient. Formalin-inactivated vaccine candidates were formulated at various doses [low dose (LD), 5 μg; medium dose (MD), 10 μg; high dose (HD), 20 μg)] and mixed with an alum adjuvant. BALB/c mice received the same doses of the vaccine formulations three times at 3-week intervals. The humoral endpoint IgG responses were evaluated and compared for the MBVax and CCVax treatments. The duration of the presence of IgG and neutralizing antibody (Ab) titers was evaluated and compared until up to 1 year after immunization. The humoral IgG responses indicated that the CCVax and MBVax candidates enhanced the IgG endpoint titers in a dose-dependent manner, which were induced more strongly in all the CCVax groups than in the MBVax mice. The fold changes in neutralizing Ab levels were also found to be higher in the CCVax groups: between 2- and 7.6-fold after the second week of the last immunization. The neutralization titers peaked 4 months after immunization in all the vaccine-receiving groups, but these were still comparable at the end of the first year. The CCVax formulations induced higher IgG and neutralizing Ab titers at all the measured time points. In this study, we showed that cell-culture-purified and formalin-inactivated vaccine candidates induced strong and robust immunity in vaccinated mice dose-dependently, more so than mouse-brain-derived vaccines.

Activation and Kinetics of Circulating T Follicular Helper Cells, Specific Plasmablast Response, and Development of Neutralizing Antibodies following Yellow Fever Virus Vaccination.

A single dose of the replication-competent, live-attenuated yellow fever virus (YFV) 17D vaccine provides lifelong immunity against human YFV infection. The magnitude, kinetics, and specificity of B cell responses to YFV 17D are relatively less understood than T cell responses. In this clinical study, we focused on early immune events critical for the development of humoral immunity to YFV 17D vaccination in 24 study subjects. More specifically, we studied the dynamics of several immune cell populations over time and the development of neutralizing Abs. At 7 d following vaccination, YFV RNA in serum as well as several antiviral proteins were detected as a sign of YFV 17D replication. Activation of Th1-polarized circulating T follicular helper cells followed germinal center activity, the latter assessed by the surrogate marker CXCL13 in serum. This coincided with a plasmablast expansion peaking at day 14 before returning to baseline levels at day 28. FluoroSpot-based analysis confirmed that plasmablasts were specific to the YFV-E protein. The frequencies of plasmablasts correlated with the magnitude of neutralizing Ab titers measured at day 90, suggesting that this transient B cell subset could be used as an early marker of induction of protective immunity. Additionally, YFV-specific memory B cells were readily detectable at 28 and 90 d following vaccination, and all study subjects tested developed protective neutralizing Ab titers. Taken together, these studies provide insights into key immune events leading to human B cell immunity following vaccination with the YFV 17D vaccine.

Differential durability of immune responses to measles and mumps following MMR vaccination

The development and wide-spread use of mumps vaccine resulted in a dramatic and sustained decrease in the incidence of mumps disease; however, since 2000, an increase in the size and number of mumps outbreaks in the United States and other countries has sparked renewed interest in the durability of mumps-specific immunity elicited by mumps vaccination. The most likely explanation for mumps cases in previously immunized persons may be secondary vaccine failure, or waning immunity. In the current study, we examined changes in markers of measles and mumps immunity at two timepoints, approximately 7 and 17 years after two-dose MMR-II® vaccination, in a cohort of 98 healthy adults.Our results indicate that mumps IgG titers exhibited a large and significant decline during this time period, while mumps neutralizing Ab titers were relatively stable. There was a similar discrepancy with measles-specific immune responses. For both pathogens, neutralizing antibody titers were fairly low and, given the length of time since vaccination, may have already declined. These data suggest that specific immune outcomes may wane at different rates and highlight our currently incomplete understanding of protective immune responses to mumps and measles.

Interface between Maternal Antibodies and Natural Challenge for Bovine Viral Diarrhea Virus (BVDV) in Holstein Heifers

Background: Newborn calves are agammaglobulinemic, immunosuppressed and immunologically immature at birth. The passive immune transfer is fundamental to protect calves against pathogens. The decay of maternal antibodies precedes the immune maturation at puberty enhancing the susceptibility of calves to infections caused by BVDV. Then, the objective of this research was to evaluate the interface between passive and active immunity for Bovine Viral Diarrhea Virus (BVDV) in Holstein dairy heifers in the first 13 months of age to detect susceptibility periods and establish prophylactic measures on prevention of Bovine Viral Diarrhea.Materials, Methods &amp; Results: Sera were analyzed from 585 heifers by serum neutralization (SN) and enzyme linked immunosorbent assay (ELISA) for the p80 protein of BVDV. Heifers were categorized according to their age by the month of life. Heifers were seropositive (100%) from 1st to 13th. Median of neutralizing antibodies (Ab) titers obtained from 1st up to 13th month were 316.2; 125.9; 63.1; 50.1; 50.1; 39.8; 63.1; 63.1; 39.8; 79.4, 100.0; 74.4; and 79.4, respectively. The neutralizing Ab titers obtained in 1st month were different of the values observed from 2nd until 13th (P &lt; 0.001), furthermore the Ab titers from 2nd month was statistical different of 4th (P = 0.01) and 6th (P = 0.05). The frequencies (%) of positive heifers for p80 from 1st up to 13th were 24.7; 18.2; 10.4; 11.8; 73.3; 73.8; 72.4; 58.1; 45.9; 48.4; 46.2; 43.8 and 61.5, respectively. The correlation observed for neutralizing Ab titers and age was negative and weak (ρ= -0.299; P &lt; 0.001). On the other hand, the correlation between positive heifers for p80 and age was positive and moderate (ρ= 0.319; P &lt; 0.001).Discussion: The newborn calves had higher titers of neutralizing antibodies than other age groups and some calves were seropositive for the p80 protein. This profile points to the transfer of maternal antibodies produced by vaccination and/or natural exposure to BVDV. The exposure of the cows to the inactivated and live virus stimulates the production of neutralizing antibodies to the structural proteins of the virus, particularly the glycoprotein E2, detected by the serum neutralization test. The titers of serum neutralizing antibodies and the frequencies of seropositive for p80 protein decreased gradually from the first to the 4-6th month of life due to the metabolization of maternal immunoglobulins acquired by ingestion of colostrum. Frequencies of seropositive animals for protein p80 increased from the fifth month of life, which is the same moment that was observed declined of neutralizing maternal antibody titers. The phase of higher frequency of p80 positive animals coincides with greater rates of Babesiosis and Anaplasmosis. This history could justify the importance of the BVDV immunodepression as a risk factor for concomitant diseases. In general, the neutralizing antibody titers increased after the peaks of positive reactions to the p80 protein, but this moment coincides with the primo-vaccination in calves. Therefore, it is not possible to state the origin of these antibodies. Correlations between ages and serologic tests are consistent with previous data reporting the decrease in antibody titers and increase of seropositive animals for p80 protein, from the first month of life to puberty. In conclusion, maternal neutralizing Ab titers had gradual decreased whereas the frequency of positive heifers for p80 had increased values. The inversion observed between the maternal antibody titers and the increase in antibody for p80 indicates the moment of greatest risk for natural infections caused by BVDV.

Intranasal Inactivated Influenza Vaccines: a Reasonable Approach to Improve the Efficacy of Influenza Vaccine?

Influenza is a contagious, acute respiratory disease caused by the influenza virus. The mucosal lining in the host respiratory tract is not only the site of virus infection, but also the site of defense; it is at this site that the host immune response targets the virus and protects against reinfection. One of the most effective methods to prevent influenza is to induce specific antibody (Ab) responses in the respiratory tract by vaccination. Two types of influenza vaccines, intranasal live attenuated influenza virus (LAIV) vaccines and parenteral (injectable) inactivated vaccines, are currently used worldwide. These vaccines are approved by the European Medicines Agency (EMA) and the US Food and Drug Administration. Live attenuated vaccines induce both secretory IgA (S-IgA) and serum IgG antibodies (Abs), whereas parenteral vaccines induce only serum IgG Abs. However, intranasal administration of inactivated vaccines together with an appropriate adjuvant induces both S-IgA and IgG Abs. Several preclinical studies on adjuvant-combined, nasal-inactivated vaccines revealed that nasal S-IgA Abs, a major immune component in the upper respiratory tract, reacted with homologous virus hemagglutinin (HA) and were highly cross-reactive with viral HA variants, resulting in protection and cross-protection against infection by both homologous and variant viruses, respectively. Serum-derived IgG Abs, which are present mainly in the lower respiratory tract, are less cross-reactive and cross-protective. In addition, our own clinical trials have shown that nasal-inactivated whole virus vaccines, including a built-in adjuvant (single-stranded RNA), induced serum hemagglutination inhibition (HI) Ab titers that fulfilled the EMA criteria for vaccine efficacy. The nasal-inactivated whole virus vaccines also induced high levels of nasal HI and neutralizing Ab titers, although we have not yet evaluated the nasal HI titers due to the lack of official criteria to establish efficacy based on this parameter. Data suggest that adjuvant-combined nasal-inactivated vaccines have advantages over the current injectable vaccine because the former induce both S-IgA and serum IgG Abs. In addition, nasal-inactivated vaccines seem to be superior to the LAIV vaccines, because non-infectious preparations could be used in high-risk groups. Thus, the development of intranasal inactivated vaccines is recommended, because such vaccines are expected to improve the efficacy of influenza vaccines.

A novel, live-attenuated vesicular stomatitis virus vector displaying conformationally intact, functional HIV-1 envelope trimers that elicits potent cellular and humoral responses in mice.

Though vaccination with live-attenuated SIV provides the greatest protection from progressive disease caused by SIV challenge in rhesus macaques, attenuated HIV presents safety concerns as a vaccine; therefore, live viral vectors carrying HIV immunogens must be considered. We have designed a replication-competent vesicular stomatitis virus (VSV) displaying immunogenic HIV-1 Env trimers and attenuating quantities of the native surface glycoprotein (G). The clade B Env immunogen is an Env-VSV G hybrid (EnvG) in which the transmembrane and cytoplasmic tail regions are derived from G. Relocation of the G gene to the 5′terminus of the genome and insertion of EnvG into the natural G position induced a ∼1 log reduction in surface G, significant growth attenuation compared to wild-type, and incorporation of abundant EnvG. Western blot analysis indicated that ∼75% of incorporated EnvG was a mature proteolytically processed form. Flow cytometry showed that surface EnvG bound various conformationally- and trimer-specific antibodies (Abs), and in-vitro growth assays on CD4+CCR5+ cells demonstrated EnvG functionality. Neither intranasal (IN) or intramuscular (IM) administration in mice induced any observable pathology and all regimens tested generated potent Env-specific ELISA titers of 104–105, with an IM VSV prime/IN VSV boost regimen eliciting the highest binding and neutralizing Ab titers. Significant quantities of Env-specific CD4+ T cells were also detected, which were augmented as much as 70-fold by priming with IM electroporated plasmids encoding EnvG and IL-12. These data suggest that our novel vector can achieve balanced safety and immunogenicity and should be considered as an HIV vaccine platform.

Immunogenicity of a live-attenuated Japanese encephalitis vaccine in children and adolescents after hematopoietic stem cell transplantation.

There have been no recommendations for revaccination with the Japanese encephalitis (JE) vaccine in post-hematopoietic stem cell transplantation (HSCT) patients. This study aimed to measure the immunogenic response to a live-attenuated JE vaccine (SA 14-14-2) in post-HSCT patients. JE-specific neutralizing Ab titers were measured before and after the JE vaccination. The patients with Ab titers <10 at the 3-month time point received a second injection at 6 months. A total of 28 patients (male:female=11:17) with a median age of 13 years (4-21 years) were included. The underlying diseases were thalassemia (50%) and hematologic malignancies (50%). Ten patients (35.7%) had Ab titers above the preventive level before vaccination. Nine of 18 patients (50%) seroconverted at 3 months after a single JE vaccination, but only three of these patients had sustained protective Ab levels. Seven of nine patients (78%) seroconverted at 3 months after a second JE vaccine injection, and all of these patients sustained protective Ab levels at 12 months. In conclusion, post-HSCT patients had low seroconversion rates after a single dose of the live-attenuated JE vaccine. These patients may require at least two doses of the JE vaccine to ensure protective Ab levels.

A Multi-Disciplinary Approach to Identification and Treatment of Hyperglycemia on an Inpatient Hematopoietic Stem Cell Transplant (HSCT) Unit

s / Biol Blood Marrow Transplant 20 (2014) S184eS210 S199 JE vaccine (SA 14-14-2) has lesser local reaction and milder systemic side effects compared to inactivated JE vaccine. Since antibody (Ab) titers to vaccine-preventable diseases decline after hematopoietic stem cell transplantation (HSCT), therefore reimmunization recommendations have been developed for post HSCT survivors. However, there has been no recommendation for revaccination of JE vaccine in post HSCT recipients. This study aimed to measure the immunogenic response to the live-attenuated JE vaccine (SA 14-14-2) in post HSCT recipients. We enrolled patients who underwent allogeneic HSCT at least 2 years, discontinued immunosuppressive agents at least 6 months, and had no evidence of chronic GVHD. The live-attenuated JE vaccine (SA 14-14-2) was administered to the patients. JE neutralizing Ab titers were measured before JE vaccination, then at 1st, 3rd, 6th, and 12th months after vaccination by a plaque reduction neutralizing test. Patients with Ab titer less than 10 at 3rd month received a second injection at 6th month. Then, the JE titers were tested at 7th, 9th, 12th, and 18th months. Side effects of the JE vaccine were recorded by vaccine card supplied to parents or guardians. A total of 28 patients (M:F 1⁄4 11:17) with a median age of 13 years (4-21 years) participated in the study. The median time from HSCT was 4.13 years (2.1-9.8 years). The underlying diseases were thalassemia (50%) and hematologic malignancies (50%). Ten patients (36%) had Ab in the preventive range before vaccination (group 1). Nine of 18 patients (50%) seroconverted at 3rd month after single JE vaccination (group 2) but only 3 of them had sustained protective Ab levels at 12th month. Nine patients remained absence of JE Ab after the 1st injection (group 3). Seven of these 9 patients (78%) seroconverted at 3rd month after 2nd JE vaccine injection which all of them could sustain the protective Ab levels at 12th month. There was no difference of lymphocyte subset (CD4, 8, 16/56, and 19) between these 3 groups. There was no incidence of systemic reaction reported in this cohort. In conclusion, post HSCT survivors had low seroconversion rate after single dose of the live-attenuated JE vaccine (SA 14-142). Post HSCT survivors living in or traveling to the JE endemic countries may require at least 2 doses of the JE vaccine to ensure the protective Ab level.

Live-virus exposure of vaccine-protected macaques alters the anti-HIV-1 antibody repertoire in the absence of viremia

BackgroundWe addressed the question whether live-virus challenges could alter vaccine-induced antibody (Ab) responses in vaccinated rhesus macaques (RMs) that completely resisted repeated exposures to R5-tropic simian-human immunodeficiency viruses encoding heterologous HIV clade C envelopes (SHIV-Cs).ResultsWe examined the Ab responses in aviremic RMs that had been immunized with a multi-component protein vaccine (multimeric HIV-1 gp160, HIV-1 Tat and SIV Gag-Pol particles) and compared anti-Env plasma Ab titers before and after repeated live-virus exposures. Although no viremia was ever detected in these animals, they showed significant increases in anti-gp140 Ab titers after they had encountered live SHIVs. When we investigated the dynamics of anti-Env Ab titers during the immunization and challenge phases further, we detected the expected, vaccine-induced increases of Ab responses about two weeks after the last protein immunization. Remarkably, these titers kept rising during the repeated virus challenges, although no viremia resulted. In contrast, in vaccinated RMs that were not exposed to virus, anti-gp140 Ab titers declined after the peak seen two weeks after the last immunization. These data suggest boosting of pre-existing, vaccine-induced Ab responses as a consequence of repeated live-virus exposures. Next, we screened polyclonal plasma samples from two of the completely protected vaccinees by peptide phage display and designed a strategy that selects for recombinant phages recognized only by Abs present after – but not before – any SHIV challenge. With this “subtractive biopanning” approach, we isolated V3 mimotopes that were only recognized after the animals had been exposed to live virus. By detailed epitope mapping of such anti-V3 Ab responses, we showed that the challenges not only boosted pre-existing binding and neutralizing Ab titers, but also induced Abs targeting neo-antigens presented by the heterologous challenge virus.ConclusionsAnti-Env Ab responses induced by recombinant protein vaccination were altered by the multiple, live SHIV challenges in vaccinees that had no detectable viral loads. These data may have implications for the interpretation of “vaccine only” responses in clinical vaccine trials.

Anti-erythropoietin and anti-thrombopoietin antibodies induced after administration of recombinant human erythropoietin

Recombinant human erythropoietin (rhEPO) has been successfully used for correcting renal anemia. However, recent studies have raised some concerns about the safety of rhEPO treatment due to its immunogenic side effect — pure red cell aplasia (PRCA). We now report a case of development of anti-EPO neutralizing antibodies (Abs) implicated in thrombocytopenia as well as erythrocytopenia. A 35-year-old man had a history of administering rhEPO (epoetin alfa, epoetin beta and darbepoetin alfa) for 2years to treat renal anemia. The hematological parameters were collected. Anti-EPO, anti-platelet, and anti-thrombopoietin (TPO) Ab assays were performed to test the presence of autoreactive Abs. After performing antibody assays due to severe resistance to rhEPO treatment, a high titer of anti-EPO neutralizing Abs was detected. However, unexpectedly, this patient also showed thrombocytopenia rather than PRCA. We investigated the cause of the marked thrombocytopenia and found anti-TPO Abs in patient serum. To our best knowledge, this is the first report of the development of anti-TPO Abs during rhEPO treatment for anemia.

Immunization with Wild-Type or CD4-Binding-Defective HIV-1 Env Trimers Reduces Viremia Equivalently following Heterologous Challenge with Simian-Human Immunodeficiency Virus

We recently reported that rhesus macaques inoculated with CD4-binding-competent and CD4-binding-defective soluble YU2-derived HIV-1 envelope glycoprotein (Env) trimers in adjuvant generate comparable levels of Env-specific binding antibodies (Abs) and T cell responses. We also showed that Abs directed against the Env coreceptor binding site (CoRbs) were elicited only in animals immunized with CD4-binding-competent trimers and not in animals immunized with CD4-binding-defective trimers, indicating that a direct interaction between Env and CD4 occurs in vivo. To investigate both the overall consequences of in vivo Env-CD4 interactions and the elicitation of CoRbs-directed Abs for protection against heterologous simian-human immunodeficiency virus (SHIV) challenge, we exposed rhesus macaques immunized with CD4-binding-competent and CD4-binding-defective trimers to the CCR5-tropic SHIV-SF162P4 challenge virus. Compared to unvaccinated controls, all vaccinated animals displayed improved control of plasma viremia, independent of the presence or absence of CoRbs-directed Abs prior to challenge. Immunization resulted in plasma responses that neutralized the heterologous SHIV challenge stock in vitro, with similar neutralizing Ab titers elicited by the CD4-binding-competent and CD4-binding-defective trimers. The neutralizing responses against both the SHIV-SF162P4 stock and a recombinant virus pseudotyped with a cloned SHIV-SF162P4-derived Env were significantly boosted by the SHIV challenge. Collectively, these results suggest that the capacity of soluble Env trimers to interact with primate CD4 in vivo and to stimulate the production of moderate titers of CoRbs-directed Abs did not influence the magnitude of the neutralizing Ab recall response after viral challenge or the subsequent control of viremia in this heterologous SHIV challenge model.

CD1d-Dependent B-Cell Help by NK-Like T Cells Leads to Enhanced and Sustained Production of Bacillus anthracis Lethal Toxin-Neutralizing Antibodies

The current Bacillus anthracis vaccine consists largely of protective antigen (PA), the protein of anthrax toxin that mediates entry of edema factor (EF) or lethal factor (LF) into cells. PA induces protective antibody (Ab)-mediated immunity against Bacillus anthracis but has limited efficacy and duration. We previously demonstrated that activation of CD1d-restricted natural killer-like T cells (NKT) with a CD1d-binding glycolipid led to enhanced Ab titers specific for foreign antigen (Ag). We therefore tested the hypothesis that activation of NKT cells with the CD1d ligand (alpha-galactosylceramide [alpha-GC]) at the time of immunization improves PA-specific Ab responses. We observed that alpha-GC enhanced PA-specific Ab titers in C57BL/6 mice. In CD1d(-/-) mice deficient in type I and type II NKT cells the anti-PA Ab response was diminished. In Jalpha281(-/-) mice expressing CD1d but lacking type I alpha-GC-reactive NKT cells, alpha-GC did not enhance the Ab response. In vitro neutralization assays were performed and showed that the Ab titers correlated with protection of macrophages against anthrax lethal toxin (LT). The neutralization capacity of the Ab was further tested in lethal challenge studies, which revealed that NKT activation leads to enhanced in vivo protection against LT. Anti-PA Ab titers, neutralization, and protection were then measured over a period of several months, and this revealed that NKT activation leads to a sustained protective Ab response. These results suggest that NKT-activating CD1d ligands could be exploited for the development of improved vaccines for Bacillus anthracis that increase not only neutralizing Ab titers but also the duration of the protection afforded by Ab.

Gene Expression Patterns Induced by HPV-16 L1 Virus-Like Particles in Leukocytes from Vaccine Recipients

Human papillomavirus (HPV) virus-like particle (VLP) vaccines were recently licensed. Although neutralizing Ab titers are thought to be the main effectors of protection against infection, early predictors of long-term efficacy are not yet defined and a comprehensive understanding of innate and adaptive immune responses to vaccination is still lacking. Here, microarrays were used to compare the gene expression signature in HPV-16 L1 VLP-stimulated PBMCs from 17 vaccine and 4 placebo recipients before vaccination and 1 mo after receiving the second immunization. Vaccination with a monovalent HPV-16 L1 VLP vaccine was associated with modulation of genes involved in the inflammatory/defense response, cytokine, IFN, and cell cycle pathways in VLP-stimulated PBMCs. Additionally, there was up-regulation of probesets associated with cytotoxic (GZMB, TNFSF10) and regulatory (INDO, CTLA4) activities. The strongest correlations with neutralizing Ab titers were found for cyclin D2 (CCND2) and galectin (LGALS2). Twenty-two differentially expressed probesets were selected for confirmation by RT-PCR in an independent sample set. Agreement with microarray data was seen for more than two-thirds of these probesets. Up-regulation of immune/defense response genes by HPV-16 L1 VLP, in particular, IFN-induced genes, was observed in PBMCs collected before vaccination, with many of these genes being further induced following vaccination. In conclusion, we identified important innate and adaptive response-related genes induced by vaccination with HPV-16 L1 VLP. Further studies are needed to identify gene expression signatures of immunogenicity and long-term protection with potential utility in prediction of long-term HPV vaccination outcomes in clinical trials.