Strain-specific Antibodies Research Articles

Optimizing immunization protocols to elicit broadly neutralizing antibodies

Natural infections and vaccination with a pathogen typically stimulate the production of potent antibodies specific for the pathogen through a Darwinian evolutionary process known as affinity maturation. Such antibodies provide protection against reinfection by the same strain of a pathogen. A highly mutable virus, like HIV or influenza, evades recognition by these strain-specific antibodies via the emergence of new mutant strains. A vaccine that elicits antibodies that can bind to many diverse strains of the virus-known as broadly neutralizing antibodies (bnAbs)-could protect against highly mutable pathogens. Despite much work, the mechanisms by which bnAbs emerge remain uncertain. Using a computational model of affinity maturation, we studied a wide variety of vaccination strategies. Our results suggest that an effective strategy to maximize bnAb evolution is through a sequential immunization protocol, wherein each new immunization optimally increases the pressure on the immune system to target conserved antigenic sites, thus conferring breadth. We describe the mechanisms underlying why sequentially driving the immune system increasingly further from steady state, in an optimal fashion, is effective. The optimal protocol allows many evolving B cells to become bnAbs via diverse evolutionary paths.

Limited Tryptic Digestion-Isotope Dilution Mass Spectrometry (LTD-IDMS): A Reagent-Free Analytical Assay To Quantify Hemagglutinin of A(H5N1) Vaccine Material.

Avian influenza viruses, such as A(H5N1) and A(H7N9), are primary public health concerns due to their pandemic potential. Influenza vaccines represent the most effective response to this threat especially with timely provision. The current pandemic response timelines require a substantial period for strain-specific reference antigen and sera preparation for use with single-radial immunodiffusion (SRID), the accepted vaccine potency assay. To address this time lag, the isotope dilution mass spectrometry (IDMS) method was developed to quantify the absolute hemagglutinin (HA, the main influenza antigen) amount in the vaccine without the need for purified, inactivated, and calibrated virus reference antigens. However, an additional challenge in determining potency is to differentiate between vaccine antigens in their most potent form from other less potent, stressed antigen forms. The limited trypsin digestion (LTD) method has been developed and does not require strain-specific full-length reference antigens or antibodies; instead, stressed HA is selectively degraded, leaving the more potent form to be measured. LTD, followed by precipitation and IDMS, allows for efficient differentiation between potent and significantly less potent HA for vaccine release and potency testing across the vaccine's shelf life. In this study, we tested the LTD-IDMS assay on A(H5N1) vaccine material that had been stressed by low pH, heat, and multiple freeze-thaw cycles. The results showed that the LTD-IDMS method effectively quantified the potent HA in A(H5N1) vaccine material with results comparable to SRID. As such, it shows great promise to complement and potentially replace SRID in a pandemic when strain-specific reagents may not be readily available.

Donor's graft ex vivo T-cell depletion with fludarabine reduces graft-versus-host disease signs and improves survival after intestinal transplantation - an experimental study.

Intestinal passenger T leukocytes are responsible for graft-versus-host disease (GvHD) in intestinal transplantation (ITx). We hypothesized that ex vivo fludarabine treatment of the bowel graft would diminish the risk of GvHD and improve overall survival post-transplant. We performed isolated heterotopic small bowel transplantations from Lewis (LEW) to Brown Norway (BN) rat strains, which generated GvHD signs from the fourth day post-transplant. These symptoms included rash, weight loss, piloerection, and diarrhea. The grafts of one of the experimental groups were immersed and sealed in cold Celsior preservation solution with 1000µm fludarabine for 1h, prior to its implantation into recipient animals. No histological signs of intestinal tissue alterations were observed after fludarabine treatment. Fludarabine-treated bowel recipients showed significantly later and milder clinical signs of GvHD and reduced total donor cell chimerism, as determined by flow cytometry using strain-specific anti-HLA antibodies. Additionally, fludarabine treatment prolonged recipients' overall survival (13.5days±0.3days vs. 9.2days±0.5). We conclude that active modification of the intestinal leukocyte composition is advantageous in our ITx animal model. Immunosuppression with fludarabine during the surgical procedure, which could be translated directly to the clinic, protects bowel recipients from GvHD and improves overall post-transplant survival.

ELISA-Based Assay for Studying Major and Minor Group Rhinovirus-Receptor Interactions.

Rhinovirus (RV) infections are a major cause of recurrent common colds and trigger severe exacerbations of chronic respiratory diseases. Major challenges for the development of vaccines for RV include the virus occurring in the form of approximately 160 different serotypes, using different receptors, and the need for preclinical models for the screening of vaccine candidates and antiviral compounds. We report the establishment and characterization of an ELISA-based assay for studying major and minor group RV–receptor interactions. This assay is based on the interaction of purified virus with plate-bound human receptor proteins, intercellular adhesion molecule 1 (ICAM-1), and low density lipoprotein receptor (LDLR). Using RV strain-specific antibodies, we demonstrate the specific binding of a panel of major and minor RV group types including RV-A and RV-B strains to ICAM-1 and LDLR, respectively. We show that the RV–receptor interaction can be blocked with receptor-specific antibodies as well as with soluble receptors and neutralizing RV-specific antibodies. The assay is more sensitive than a cell culture-based virus neutralization test. The ELISA assay will therefore be useful for the preclinical evaluation for preventive and therapeutic strategies targeting the RV–receptor interaction, such as vaccines, antibodies, and anti-viral compounds.

Immune tolerance limits effective immunity to epitopes targeted by universal influenza vaccines

Abstract A universal influenza vaccine could save millions of lives in the event of a deadly pandemic. It is not clear why antibodies specific for conserved regions of influenza viruses are so rare. One possibility is that these antibodies have a higher potential to cross-react to self-proteins, and therefore B cells that generate these antibodies are deleted through tolerance mechanisms. Therefore, we examined whether cross-reactive influenza antibodies had a higher potential to be autoreactive than antibodies specific for one subtype of influenza. We previously demonstrated that H3N2-vaccinated mice treated with a low dose of rapamycin had more cross-reactive influenza antibodies and were better protected against subsequent lethal infections of multiple subtypes. Thus, we utilized rapamycin to increase the frequency of influenza cross-reactive antibodies, and tested whether these antibodies were more reactive to self-proteins than strain-specific antibodies. We found that mice with increased levels of cross-reactive influenza antibodies also had more IgM antibodies specific for self-antigens. Importantly, the increase in autoreactive IgM antibodies correlated with increased susceptibility to disease in mouse models of multiple sclerosis, lupus, and Guillian-Barre Syndrome. Together, our results suggest that influenza cross-reactive antibodies have the potential to be autoreactive. These data have important implications for developing universal influenza vaccines designed to generate durable influenza cross-reactive antibodies.

Rational Vaccine Design in the Time of COVID-19

As scientists consider SARS-CoV-2 vaccine design, we discuss problems that may be encountered and how to tackle them by what we term “rational vaccine design.” We further discuss approaches to pan-coronavirus vaccines. We draw on experiences from recent research on several viruses including HIV and influenza, as well as coronaviruses.

Microarray analyses reveal strain-specific antibody responses to Plasmodium falciparum apical membrane antigen 1 variants following natural infection and vaccination

Vaccines based on Plasmodium falciparum apical membrane antigen 1 (AMA1) have failed due to extensive polymorphism in AMA1. To assess the strain-specificity of antibody responses to malaria infection and AMA1 vaccination, we designed protein and peptide microarrays representing hundreds of unique AMA1 variants. Following clinical malaria episodes, children had short-lived, sequence-independent increases in average whole-protein seroreactivity, as well as strain-specific responses to peptides representing diverse epitopes. Vaccination resulted in dramatically increased seroreactivity to all 263 AMA1 whole-protein variants. High-density peptide analysis revealed that vaccinated children had increases in seroreactivity to four distinct epitopes that exceeded responses to natural infection. A single amino acid change was critical to seroreactivity to peptides in a region of AMA1 associated with strain-specific vaccine efficacy. Antibody measurements using whole antigens may be biased towards conserved, immunodominant epitopes. Peptide microarrays may help to identify immunogenic epitopes, define correlates of vaccine protection, and measure strain-specific vaccine-induced antibodies.

Is there antibody-dependent enhancement in SARS Coronavirus 2?

Dear Editor, As primary care physicians across the world cater to ongoing outbreak due to COVID-19 pandemic, it is perplexing to see the geographical discrepancy in the number of severe cases as well as deaths across countries. Although there are other significant factors, we raise a pertinent question, whether antibody-dependent enhancement (ADE) is a possible contributing factor. When a virus enters the host, several types of antibodies are formed. Some of these antibodies are neutralising, which hinders the replication of the virus by inhibiting essential steps such as the interaction of the virus with the host cell receptor. In some viruses, such as flaviviruses, enhancing antibodies are being described, which may facilitate the entry of the virus into the host cells. This immune-mediated enhancement of entry and pathogenesis is termed as ADE.[1] ADE can be mediated by Fc receptors or complement receptors.[1] Besides, ADE has been associated with increased severity. Such an effect has been well-characterised with dengue, where antibody to one serotype enhances the severity of infection due to another serotype. In coronaviruses, it has been suggested that the severity of severe acute respiratory syndrome (SARS) could have been partly due to ADE to other human coronaviruses such as 229E. In-vitro studies have shown that antibodies against SARS-CoV-1 at high dilutions resulted in ADE. However, ADE could not be demonstrated in vaccine models of rhesus macaques. Reports of ADE have also been described for the Middle East respiratory syndrome as well.[234] In COVID-19, several reasons, such as higher median age has been put forward to explain a higher percentage of severity and mortality in certain regions. Whether ADE can serve as a possible explanation for the uneven geographic distribution of severity and mortality remains to be seen. Individuals in these areas may have been primed with pre-existing locally circulating human coronaviruses and ADE to these coronaviruses may have some role in the increased severity of COVID-19. The prevalence of four human coronaviruses (HCoV-OC43, 229E, NL63 and HKU1) before COVID-19 outbreak in regions with high reported mortality are as follows: 2.4% (Spain), 5.5% (Iran), 9.3% (France), Italy (10.5%) and China (23%).[56789] There is a need for serological surveys of human coronaviruses in serum samples of COVID-19 affected patients (mild and severe) to understand whether there is a correlation between pre-existing strain-specific antibodies to locally circulating human coronaviruses and severity of COVID 19. Convalescent plasma therapy from patients who have recovered from COVID-19 has been tried in several case series as a treatment with good results.[10] Vaccine trials across the world will start in the coming months. Besides, understanding its possible role in severity, knowledge of ADE will be helpful for the primary care physicians to understand the safety and efficacy of convalescent plasma therapy and vaccines for COVID-19. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Influenza Vaccination: Accelerating the Process for New Vaccine Development in Older Adults.

Increased susceptibility to the serious complications of influenza is common in older adults. It is often ascribed to weakening of the immune system with age, and 90% of influenza-related deaths occur in older adults despite widespread vaccination programs. Common chronic conditions not only contribute to the loss of immune protection after vaccination and increase the risk for serious outcomes of influenza, but also increase the long-term consequences following hospitalization. Interactions of T and B cell ageing, chronic elevation of inflammatory cytokines (sometimes dubbed "inflammaging"), and dysregulated acute cytokine production pose major challenges to the development of new and more effective vaccines. However, these age-related problems are modifiable, as we have shown, and provide a clear margin for improvement. This chapter describes how an exclusive focus on developing influenza vaccines to stimulate strain-specific antibody responses against the hemagglutinin surface glycoprotein of the influenza virus, to the exclusion of other potentially important mechanisms, is missing the mark in terms of preventing the serious complications of influenza in older adults. Novel approaches are needed to enhance antibody-mediated protection against infection and stimulate cell-mediated immune responses to clear influenza virus from the lungs. These strategies for improving vaccine effectiveness will address the public health need for "vaccine prevention of disability" to mitigate the global pressures of aging populations on health and social care systems.

Heterogeneity of human serum antibody responses to P. gingivalis in periodontitis: Effects of age, race/ethnicity, and sex

Aging humans display an increased prevalence and severity of periodontitis, although the mechanisms underlying these findings remain poorly understood. This report examined antigenic diversity of P. gingivalis related to disease presence and patient demographics. Serum IgG antibody to P. gingivalis strains ATCC33277, FDC381, W50 (ATCC53978), W83, A7A1-28 (ATCC53977) and A7436 was measured in 426 participants [periodontally healthy (n = 61), gingivitis (N = 66) or various levels of periodontitis (N = 299)]. We hypothesized that antigenic diversity in P. gingivalis could contribute to a lack of “immunity” in the chronic infections of periodontal disease. Across the strains, the antibody levels in the oldest age group were lower than in the youngest groups, and severe periodontitis patients did not show higher antibody with aging. While 80 % of the periodontitis patients in any age group showed an elevated response to at least one of the P. gingivalis strains, the patterns of individual responses in the older group were also substantially different than the other age groups. Significantly greater numbers of older patients showed strain-specific antibody profiles to only 1 strain. The findings support that P. gingivalis may demonstrate antigenic diversity/drift within patients and could be one factor to help explain the inefficiency/ineffectiveness of the adaptive immune response in managing the infection.

Antibody-dependent cell-mediated cytotoxicity antibody responses to inactivated and live-attenuated influenza vaccination in children during 2014–15

BackgroundSeasonal influenza vaccines aim to induce strain-specific neutralizing antibodies. Non-neutralizing antibodies may be more broadly cross-reactive and still protect through mechanisms including antibody-dependent cell-mediated cytotoxicity (ADCC). Influenza vaccines may stimulate ADCC antibodies in adults, but whether they do so in children is unknown. Here we examined how vaccination affects cross-reactive ADCC antibody responses in children after receipt of inactivated trivalent vaccine (IIV3) or quadrivalent live-attenuated vaccine (LAIV4). MethodsChildren aged 5–17 were recruited in fall 2014 to provide pre- and post-vaccination serum samples. Children aged 5–9 received LAIV4 based on then-current recommendation, and older children were randomly assigned to IIV3 or LAIV4. We used microtiter-plate-based flow cytometry with an NK cell line to examine ADCC antibody responses to the 2014–15 H3N2 vaccine component (A/Texas/50/2012 [TX12]) and a drifted strain, A/Switzerland/9715293/2013 (SW13). Responses were stratified by two-season (2013–14 and 2014–15) vaccine sequence. ResultsEighty-five children received LAIV4 and 45 received IIV3. Prevaccination ADCC activity was highest in children who had received any vaccine in the prior season. Increase in ADCC antibody responses against the vaccine strain TX12 following vaccination was greatest for participants who received IIV3 in 2014–15 and LAIV4 in the prior season (geometric mean fold rise [MFR] = 1.6, 95% CI.1.23–2.11). This group also had a detectable ADCC response to the drifted SW13 strain. There was a modest ADCC response against SW13 in LAIV4 recipients who were unvaccinated in the previous season (MFR = 1.18, 95% CI 1.10–1.25). There were no significant changes in 2014–15 ADCC response to vaccination among children who had received IIV3 in 2013–14. ConclusionsVaccinating children with IIV3 after prior receipt of LAIV4 generated a modest increase in ADCC antibodies, including some cross-reactivity with an emerging drift variant. Other vaccine-induced ADCC responses were minimal and not affected by vaccine type or sequence.

Maternal Antibodies Provide Bank Voles with Strain-Specific Protection against Infection by the Lyme Disease Pathogen.

Multistrain microbial pathogens often induce strain-specific antibody responses in their vertebrate hosts. Mothers can transmit antibodies to their offspring, which can provide short-term, strain-specific protection against infection. Few experimental studies have investigated this phenomenon for multiple strains of zoonotic pathogens occurring in wildlife reservoir hosts. The tick-borne bacterium Borrelia afzelii causes Lyme disease in Europe and consists of multiple strains that cycle between the tick vector (Ixodes ricinus) and vertebrate hosts, such as the bank vole (Myodes glareolus). We used a controlled experiment to show that female bank voles infected with B. afzelii via tick bite transmit protective antibodies to their offspring. To test the specificity of protection, the offspring were challenged using a natural tick bite challenge with either the maternal strain to which the mothers had been exposed or a different strain. The maternal antibodies protected the offspring against a homologous infectious challenge but not against a heterologous infectious challenge. The offspring from the uninfected control mothers were equally susceptible to both strains. Borrelia outer surface protein C (OspC) is an antigen that is known to induce strain-specific immunity. Maternal antibodies in the offspring reacted more strongly with homologous than with heterologous recombinant OspC, but other antigens may also mediate strain-specific immunity. Our study shows that maternal antibodies provide strain-specific protection against B. afzelii in an ecologically important rodent reservoir host. The transmission of maternal antibodies may have important consequences for the epidemiology of multistrain pathogens in nature.IMPORTANCE Many microbial pathogen populations consist of multiple strains that induce strain-specific antibody responses in their vertebrate hosts. Females can transmit these antibodies to their offspring, thereby providing them with short-term strain-specific protection against microbial pathogens. We investigated this phenomenon using multiple strains of the tick-borne microbial pathogen Borrelia afzelii and its natural rodent reservoir host, the bank vole, as a model system. We found that female bank voles infected with B. afzelii transmitted to their offspring maternal antibodies that provided highly efficient but strain-specific protection against a natural tick bite challenge. The transgenerational transfer of antibodies could be a mechanism that maintains the high strain diversity of this tick-borne pathogen in nature.

EFFECT OF INFLUENZA VACCINE ON TWEAK LEVELS IN THE ELDERLY: IMPLICATION IN CARDIOVASCULAR PROTECTION

Substantial evidence suggests a protective effect of annual influenza immunization on cardiovascular diseases (CVD). The inflammatory mediator TNF-related weak inducer of apoptosis (TWEAK) is thought to be involved in the pathogenesis of CVD. We previously showed that administration of a standard-dose influenza vaccine reduced circulating TWEAK levels. This study aimed to test the hypothesis that a high-dose vaccine would have greater impact on TWEAK levels than the standard-dose. Two groups of participants matched on age and sex were included in the study. One group (n=25) received standard-dose vaccine during 2008-2009 season, the other group (n=25) received high-dose vaccine during 2014-2015 season. Soluble TWEAK (sTWEAK) levels were assessed using ELISA in serum samples collected immediately before vaccination and during the 4th week after vaccination. Vaccine-induced strain specific antibody titers were measured by hemagglutination inhibition assay. The participants had a mean age 86 years and 68% were women. Our preliminary results thus far demonstrated no statistically significant change in sTWEAK levels after vaccination in either group (Wilcoxon matched-pairs signed rank test: standard-dose group: median change [interquartile range]=11.2 [-92.2–197.1] pg/mL, p=0.72; high-dose group: -24.8 [-58.9–87.3] pg/mL, p=0.70). Pre-vaccination sTWEAK levels tended to be negatively associated with age (unadjusted linear regression of log2(x) transformed TWEAK levels on age: estimate [±SE]=-4.6% [±2.7%] change in TWEAK level per year, p=0.08). We continue to evaluate more pre- and post-vaccination samples. We have also begun exploring TWEAK expression by circulating immune cells (T and B lymphocytes and monocytes) and potential impact of influenza immunization in older adults.

A phase IIb study to determine the safety and efficacy of candidate INfluenza Vaccine MVA-NP+M1 in combination with licensed Ina CTivated infl Uenza vaccine in adult S aged 65 years and above (INVICTUS): a study protocol

Seasonal influenza has a significant annual global impact. Current influenza vaccines work by inducing strain-specific antibodies against the highly polymorphic surface proteins of the influenza virus and need to be redesigned every year, increasing their cost and limiting availability. There is a demand for a more efficacious vaccine, particularly in older adults in which the current vaccines show poor efficacy. The aim is to investigate a novel vaccine, MVA-NP+M1, which targets T cell responses to the nucleoprotein and matrix 1 core proteins of the influenza virus A, which are highly conserved, and therefore may provide long protection against a broad range of influenza strains. INVICTUS is a phase IIb study to determine the safety and efficacy of candidate INfluenza Vaccine MVA-NP+M1 in combination with licensed Ina CTivated infl Uenza vaccine in adult S aged 65 years and above is a randomised, participant-blinded, placebo-controlled, multi-centre phase IIb efficacy study planned for 2030 volunteers aged 65 and over, in primary care. The primary objective is to assess the efficacy of MVA-NP+M1 co-administered with licensed inactivated quadrivalent influenza vaccine in adults ≥65 years. Participants complete daily diaries to record solicited and unsolicited events in the first four weeks post vaccination, and influenza-like illness (ILI) symptoms and severity throughout the influenza season. We hypothesise an improvement in the primary outcome, a reduction in the average number of days spent with moderate or severe influenza-like illness during periods of influenza circulation, in the group administered with MVA-NP+M1, compared to those in the control group. Registration: ClinicalTrials.gov identifier NCT03300362. Protocol version: INVICTUS Protocol v3.0, 08 June06 2018.

A systematic review and meta-analysis of cross-reactivity of antibodies induced by H7 influenza vaccine

ABSTRACTInoculation with vaccine is the major intervention currently used to prevent influenza infections. However, it will be a challenge to produce and implement a new vaccine when a novel highly pathogenic influenza virus emerges in humans as significant infections. H7 subtype influenza viruses have similar epitopes on hemagglutinin, which can induce cross-reactive antibodies. In this study, a meta-analysis of the cross-reactivity of antibodies induced by one H7 subtype influenza vaccine against other H7 subtypes was performed. Database search was conducted in PubMed, Cochrane Library, EMBASE, MEDLINE, Chinese Biological Medicine Database (CBM), and Wanfang. A total of 9 articles comprising 811 human subjects were included in this meta-analysis. All assessed H7 influenza vaccines induced vaccine strain-specific protective antibodies [seroconversion rate (SCR) = 0.74, 95% CI (0.65, 0.82); seroprotection rate (SPR) = 0.81, 95% CI (0.78, 0.83)]. All H7 influenza virus monovalent vaccines exhibited cross-reactivity tested by hemagglutinin inhibition test (HI), microneutralization test (MN) and immunosorbent assay (ELISA) to other H7 subtype viruses. H7N1, H7N3, H7N7, and H7N9 vaccines elicited cross-reactive antibodies against other H7 subtype influenza viruses [SCR = 0.66, 95% CI (0.50, 0.82); SPR = 0.79, 95% CI (0.67, 0.91)]. The pooled SCR (95%CI) of cross-reactivity of H7N1 and H7N3 vaccines were 0.88 (0.85, 0.91) and 0.40 (0.26, 0.54), respectively. The consolidated SPR (95%CI) of H7N1 and H7N7 vaccines were 0.89 (0.86, 0.92) and 0.93 (0.81, 1.06). All H7 vaccines induced cross-reactive antibodies against H7N9 viruses [SCR = 0.69, 95% CI (0.52, 0.86); SPR = 0.85, 95% CI (0.76, 0.94)]. H7 vaccines can be used to limit influenza infection when a new highly pathogenic H7 virus appears.

Inactivation methods for whole influenza vaccine production.

Despite tremendous efforts toward vaccination, influenza remains an ongoing global threat. The induction of strain-specific neutralizing antibody responses is a common phenomenon during vaccination with the current inactivated influenza vaccines, so the protective effect of these vaccines is mostly strain-specific. There is an essential need for the development of next-generation vaccines, with a broad range of immunogenicity against antigenically drifted or shifted influenza viruses. Here, we evaluate the potential of whole inactivated vaccines, based on chemical and physical methods, as well as new approaches to generate cross-protective immune responses. We also consider the mechanisms by which some of these vaccines may induce CD8+ T-cells cross-reactivity with different strains of influenza. In this review, we have focused on conventional and novel methods for production of whole inactivated influenza vaccine. As well as chemical modification, using formaldehyde or β-propiolactone and physical manipulation by ultraviolet radiation or gamma-irradiation, novel approaches, including visible ultrashort pulsed laser, and low-energy electron irradiation are discussed. These two latter methods are considered to be attractive approaches to design more sophisticated vaccines, due to their ability to maintain most of the viral antigenic properties during inactivation and potential to produce cross-protective immunity. However, further studies are needed to validate them before they can replace traditional methods for vaccine manufacturing.

P3.60: Native spleen preservation attenuate graft versus host disease in an experimental model of modified multivisceral transplantation.

Introduction: Native spleen used to be removed in patients undergoing modified multivisceral transplantation (MMVT) increasing the risk of sepsis and graft-versus-host disease (GVHD). The mechanistic basis of these effects including mixed chimerism level is poorly understood. Based on a novel experimental procedure of MMVT that triggers GVHD, we aimed to evaluate the effects of native spleen preservation in this model in order to gain insight into the mechanisms that may mediate this phenomenon. Material and Methods: Heterotopic MMVT from Lewis to Brown Norway rats was performed. MMVT graft consisted of stomach, duodenum, pancreas, spleen and the small bowel of the donor. The native spleen was removed (MMVT-S) in half of the MMVT recipients (N=5), while the remaining preserved their own spleen (MMVT+NSP) (N=5). After MMVT mixed hematopoietic chimerism was determined in native spleen and peripheral blood samples by flow cytometry using strain-specific HLA antibodies. Also, histopathological (using Pintar score) and clinical signs of GVHD such as skin rash were evaluated. Results: all animals in the group MMVT-S presented clinical signs of GVHD such as skin rash, weight loss, and diarrhea, among others, between 7–10 days after transplantation. Skin rash was particularly remarkable in the ears and periocular area. Clinical signs of GVHD were less frequent in MMVT+NSP recipients (40%; p<0.05). Also, significant differences between groups were observed in the histopathological study and mixed chimerism in peripheral blood 3 days after MMVT (36.6 and 23.62 % in MMVT-S and MMVT+NSP respectively) (Figure 1). Also, native spleen showed an 18±5 % of chimerism (CD3+ donor cells) 1 week after transplantation. After day 10 post-MMVT, when the signs of transient GVHD disappeared, both groups presented graft rejection. Conclusions: Native spleen preservation in MMVT recipients attenuated the occurrence of GVHD and reduced the level of chimerism compared to recipients that underwent spleen removal during MMVT. Despite more studies are necessary, our preliminary results suggest that native spleen preservation increase the efficacy in removal of anti-recipient reactive clones that would explain the protective effect of native spleen preservation against GVHD.

P1.10: Donor’s graft ex vivo T cell depletion with fludarabine reduces GvHD signs and improves survival after intestine transplantation

Introduction: Intestine passenger T leukocytes are responsible of graft versus host disease (GvHD) in intestine transplantation (ITx). These lymphocytes are known to have inferior tolerogenic qualities compared with leukocytes in the liver, other solid organs and bone marrow. Fludarabine is a routinely used anti neoplasic agent with high cytotoxicity against T cells. We hypothesized that ex vivo fludarabine treatment of the bowel graft could diminish the risk of GvHD and improve post Tx overall survival. Methods: We performed isolated heterotopic ITx from Lewis (LEW) to Brown Norway (BN) rat strains. The grafts of one of the experimental groups were imbided and sealed in Celsior preserving solution with 1000 µM fludarabine during surgery (1 h), before its implantation into recipient animals. We compared a group of untreated (n = 7) vs a group of fludarabine-treated bowel recipients (n = 5). The mixed hematopoietic chimerism was determined by flow cytometry using strain-specific anti HLA antibodies. Clinical signs of GvHD or as well as post-Tx overall survival were also monitored. Results: One hour fludarabine treatment of the bowel grafts induced specific apoptosis of its passenger T cells at concentrations from 100 μM while no histological signs of intestinal tissue alterations were observed after 1000 μM fludarabine treatment. After heterotopic LEW -> BN ITx, untreated intestine recipients showed GvHD signs from the fourth day post-Tx (n = 7). These symptoms include: rash (n = 4), weight loss (n = 3), piloerection (n = 2) and diarrhea (n = 1). The chimerism or percentage of donor’s lymphocytes in the recipient rat, determined in peripheral blood, reached 6.61% (range 1.7–10.8%) at day 3 post-Tx and 2.56% (range 0–9.8%) at day 7 post-Tx (Fig. 1). Rats transplanted with a fludarabine-conditioned intestine showed statistically signifcant later and milder clinical signs of GvHD. Additionally, fludarabine treatment reduced total donor cells chimerism at day 7 and the percentage of chimeric T cells at days 3 and 7 post-Tx (Fig. 2). Both experimental groups died showing clinical signs of graft rejection. Untreated bowel grafts recipients died within 9.2 days ± 0.3 days, while fludarabine-treated graft recipients showed prolonged survival (13.5 days ± 0.3 days). Conclusions: Graft immunosupresion with fludarabine during surgical procedure protects bowel recipients of GvHD risk and improves post-Tx overall survival.

Autoreactive potential of universal influenza vaccines

Abstract A universal influenza vaccine could save millions of lives in the event of a deadly pandemic. It is not clear why antibodies specific for conserved regions of influenza viruses are so rare. One possibility is that these antibodies have a higher potential to cross-react to self-proteins, and therefore B cells that generate these antibodies are deleted through tolerance mechanisms. Therefore, we examined whether cross-reactive influenza antibodies had a higher potential to be autoreactive than antibodies specific for one subtype of influenza. We previously demonstrated that H3N2-vaccinated mice treated with a low dose of rapamycin had more cross-reactive influenza antibodies and were better protected against subsequent lethal infections of multiple subtypes. Thus, we utilized rapamycin to increase the frequency of influenza cross-reactive antibodies, and tested whether these antibodies were more reactive to self-proteins than strain-specific antibodies. We found that mice with increased levels of cross-reactive influenza antibodies also had more IgM antibodies specific for self-antigens. Although the increase in autoreactive IgM antibodies was transient, it correlated with increased susceptibility to disease in mouse models of multiple sclerosis and Guillian-Barre Syndrome. Together, our results suggest that influenza cross-reactive antibodies have the potential to be autoreactive. These data have important implications for developing universal influenza vaccines designed to generate durable influenza cross-reactive antibodies.

Immunity of a Klebsiella Pneumoniae Vaccine Independent of Antigen Specific Antibody

Abstract Objective We demonstrated the serotype independent immunity of Klebsiella pneumoniae (K. pneumoniae) vaccine. We would like to declare next if the noble effect is dependent on the antibody specific manner. Methods Mice were vaccinated via intranasal with Kp serotype 2 OMPx adjuvanted with heat-labile enterotoxin A1 domain (LTA1) and boosted at 3 weeks after the initial immunization. Protective immunity was compared among the C57Bl/6 mice, STAT3 fl/fl×CD4 Cre mice, and Il21r −/− mice by infecting with K1 strain of K. pneumoniae. Lung T-cell responses were evaluated by FACS and ELISPOT as well as K1 strain specific antibodies were assessed by ELISA. Results CD4+ T-cells of the vaccinated lungs in the CD4+ STAT3 deficient mice were significantly decreased compared to the WT mice and the Il21r −/− mice, and IL17A response by ELISPOT were significantly reduced as well. Meanwhile, K1 strain specific IgG in the serum and IgA in the lung homogenates of vaccinated WT mice were significantly increased in vaccinated mice and these responses were reduced in vaccinated CD4+ STAT3 deficient mice and Il21r −/− mice. Furthermore, vaccine induced protection was compromised in vaccinated CD4+ STAT3 deficient mice whereas the protection as well as antigen elicited lung IL-17 responses were maintained in Il21r −/− mice. Conclusions STAT3 activation in CD4+ T-cells regulated by IL-21R signaling are required for the K. pneumoniae specific antibodies, but these antibody responses were dispensable for the serotype independent immunity LTA1 adjuvanted K. pneumoniae vaccine. However, CD4 STAT3 was essential for Th17 immunity and therapeutic efficacy of the vaccine.