Pre-existing Neutralizing Antibodies Research Articles

OR23 HLA class II genes correlate with protective neutralizing antibody titers in a dengue vaccine efficacy trial

Aim A tetravalent, live attenuated dengue vaccine demonstrated efficacy, safety and immunogenicity in several clinical trials in Asia and Latin America. Efficacy differed based on infecting serotypes, presence of pre-existing dengue neutralizing antibody (NAb) titers and age. HLA class II molecules expressed on antigen presenting cells mediate CD4+ T cell stimulation of antibody production by B cells involved in vaccine-induced responses. We hypothesized that the differences in observed vaccine efficacy could be due to variation in NAb immune responses in conjunction with host HLA class II genes. Methods Samples were available from a subset of subjects that took part in the first tetravalent dengue vaccine efficacy trial conducted in Thailand. DNA was extracted from 335 saliva samples and HLA genotyping was performed using next-generation sequencing (NGS) of full-length genes. A panel of ancestry informative markers (AIMs) was genotyped to assess population stratification. Serotype-specific NAb titers were measured by plaque-reduction neutralization test 28 days after last injection. The association of NAb titers and HLA class II on dengue infection was tested by logistic regression. Linear regression was used to test association of HLA class II alleles with NAb levels after accounting for sex, age, and serotype as covariates. A minimal false discovery rate to account for multiple comparisons, with a two-sided p-value Results NGS identified 197 HLA class I and II alleles in the Thai Dengue vaccine trial. AIMs analysis did not identify population stratification comparing cases and controls. Magnitude of NAb levels post vaccination was significantly higher in the presence of HLA-DRB1*11 (p = 0.002, q = 0.08). HLA-DPB1*03:01 and *05:01 presence correlated with pre-existing NAb titers in the placebos (p = 0.005, q = 0.09; p = 0.001, q = 0.04). We did not observe a direct effect of HLA on dengue infection in either the placebo or treatment arm. Conclusions These findings suggest that specific HLA class II alleles modulate protective NAb titers in dengue infection. This is an exploratory study to identify signals to replicate in other dengue vaccine clinical studies. Understanding this HLA class II mechanism will enable improved vaccine design.

Sero-prevalence of Middle East respiratory syndrome coronavirus (MERS-CoV) specific antibodies in dromedary camels in Tabuk, Saudi Arabia.

The Middle East Respiratory Syndrome Coronavirus (MERS‐CoV) is a novel Coronavirus which was responsible of the first case of human acute respiratory syndrome in the Kingdom of Saudi Arabia (KSA), 2012. Dromedary camels are considered as potential reservoirs for the virus and seem to be the only animal host which may transmit the infection to human. Further studies are required to better understand the animal sources of zoonotic transmission route and the risks of this infection. A primary sero‐prevalence study of MERS‐CoV preexisting neutralizing antibodies in Dromedary camel serum was conducted in Tabuk, western north region of KSA, in order to assess the seopositivity of these animals and to explain their possible role in the transmission of the infection to Human. One hundred seventy one (171) serum samples were collected from healthy dromedary camels with different ages and genders in Tabuk city and tested for specific serum IgG by ELISA using the receptor‐binding S1 subunits of spike proteins of MERS‐CoV. 144 (84,21%) of the total camel sera shown the presence of protein‐specific antibodies against MERS‐CoV. These results may provide evidence that MERS‐CoV has previously infected dromedary camels in Tabuk and may support the possible role of camels in the human infection.

Screening for Neutralizing Antibodies Against Natural and Engineered AAV Capsids in Nonhuman Primate Retinas.

Adeno-associated virus (AAV) has shown promise as a therapeutic gene delivery vector for inherited retinal degenerations in both preclinical disease models and human clinical trials. The retinas of nonhuman primates (NHPs) share many anatomical similarities to humans and are an important model for evaluating AAV gene delivery. Recent evidence has shown that preexisting immunity in the form of neutralizing antibodies (NABs) in NHPs strongly correlates with weak or lack of AAV transduction in the retina when administered intravitreally, work with translational implications. This necessitates prescreening of NHPs before intravitreal delivery of AAV. In this chapter, we describe a method for screening NHP serum for preexisting NABs.

Gene Therapy for Hemophilia: Progress to Date.

Hemophilia is a congenital bleeding disorder that affects nearly half a million individuals worldwide. Joint bleeding and other co-morbidities are a significant source of debilitation for this population. Current therapies are effective but must be given lifelong at regular intervals, are costly, and are available to only about 25% of the hemophilia population living in resource-rich countries. Gene therapy for hemophilia has been in development for three decades and is now entering pivotal-stage clinical trials. While many different technology platforms exist for gene therapy, all current clinical trials for hemophilia employ adeno-associated vector (AAV)-based cell transduction. This small viral particle is capable of packaging modified F8 or F9 transgenes, can be generated robustly from cell lines, and transduces several relatively end-differentiated target tissues such as the liver with high efficiency. While pre-existing neutralizing antibodies to the AAV capsid are recognized to limit current therapy, other challenges have been identified in human studies that were not seen in preclinical studies. Both liver transaminase elevations and immune-mediated loss of transgene expression have been observed in clinical trials. Toll-like receptors, cytotoxic T cells, and other components of the immune response have been implicated in the loss of factor expression, but a full understanding of the immune response awaits clarification. Despite these challenges, many patients enrolled in gene therapy trials have attained long-term expression of factors VIII and IX. This emerging technology now represents a cure for the severe bleeding and joint damage associated with hemophilia.

Overcoming the Host Immune Response to Adeno-Associated Virus Gene Delivery Vectors: The Race Between Clearance, Tolerance, Neutralization, and Escape.

Immune responses in gene therapy with adeno-associated virus (AAV) vectors have been the object of almost two decades of study. Although preclinical models helped to define and predict certain aspects of interactions between the vector and the host immune system, most of our current knowledge has come from clinical trials. These studies have allowed development of effective interventions for modulating immunotoxicities associated with vector administration, resulting in therapeutic advances. However, the road to full understanding and effective modulation of immune responses in gene therapy is still long; the determinants of the balance between tolerance and immunogenicity in AAV vector-mediated gene transfer are not fully understood, and effective solutions for overcoming preexisting neutralizing antibodies are still lacking. However, despite these challenges, the goal of reliably delivering effective gene-based treatments is now in sight.

Application of polyploid adeno-associated virus vectors for transduction enhancement and neutralizing antibody evasion

Adeno-associated virus (AAV) vectors have been used successfully in clinical trials for patients with hemophilia or blindness, but pre-existing neutralizing antibodies (Nab) are common in the general population and exclude many patients from clinical trials. Exploration of effective strategies to enhance AAV transduction and escape from Nab activity is still imperative. Previous studies have shown the compatibility of capsids from AAV serotypes and homology of recognition sites of AAV Nab located on different capsid subunits from one virion. In this study, we co-transfected AAV2 and AAV8 helper plasmids at different ratios (3:1, 1:1 and 1:3) to assemble haploid capsids and study both their transduction efficiency and Nab escape activity. After muscular injection, all of the haploid viruses induced higher transduction than their parental AAV vectors (2- to 9-fold over AAV2), with the highest of these being the haploid vector AAV2/8 3:1. After systemic administration, a 4-fold higher transduction in the liver was observed with haploid AAV2/8 1:3 than that with AAV8 alone. We then packaged the therapeutic factor IX cassette into haploid AAV2/8 1:3 capsids and injected them into FIX knockout mice via the tail vein. Higher FIX expression and improved phenotypic correction were achieved with the haploid AAV2/8 1:3 virus vector when compared to that of AAV8. Additionally, the haploid virus AAV2/8 1:3 was able to escape AAV2 neutralization and did not increase capsid antigen presentation capacity when compared to AAV8. To improve the Nab evasion ability of the haploid virus, we produced the triploid vector AAV2/8/9 by co-transfecting AAV2, AAV8 and AAV9 helper plasmids at a ratio of 1:1:1. After systemic administration, a 2-fold higher transduction in the liver was observed with the triploid vector AAV2/8/9 than that with AAV8. Nab analysis demonstrated that the triploid AAV2/8/9 vector was able to escape Nab activity from mouse sera immunized with parental serotypes. These results indicate that polyploid viruses might potentially acquire advantages from parental serotypes for enhancement of AAV transduction and evasion of Nab recognition without increasing capsid antigen presentation in target cells. Polyploid AAV vectors can be generated from any AAV serotype, whether natural, rational, library derived or a combination thereof, providing a novel strategy that should be explored in future clinical trials in patients with neutralizing antibodies.

Structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion

Preexisting neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) pose a major, unresolved challenge that restricts patient enrollment in gene therapy clinical trials using recombinant AAV vectors. Structural studies suggest that despite a high degree of sequence variability, antibody recognition sites or antigenic hotspots on AAVs and other related parvoviruses might be evolutionarily conserved. To test this hypothesis, we developed a structure-guided evolution approach that does not require selective pressure exerted by NAbs. This strategy yielded highly divergent antigenic footprints that do not exist in natural AAV isolates. Specifically, synthetic variants obtained by evolving murine antigenic epitopes on an AAV serotype 1 capsid template can evade NAbs without compromising titer, transduction efficiency, or tissue tropism. One lead AAV variant generated by combining multiple evolved antigenic sites effectively evades polyclonal anti-AAV1 neutralizing sera from immunized mice and rhesus macaques. Furthermore, this variant displays robust immune evasion in nonhuman primate and human serum samples at dilution factors as high as 1:5, currently mandated by several clinical trials. Our results provide evidence that antibody recognition of AAV capsids is conserved across species. This approach can be applied to any AAV strain to evade NAbs in prospective patients for human gene therapy.

Neutralizing antibodies against adenovirus type 2 in normal and HIV-1-infected subjects: Implications for use of Ad2 vectors in vaccines

ABSTRACT Pre-existing neutralizing antibodies (NAbs) directed against vaccine vectors have attracted considerable research attention. Therefore, our aim was to establish a high-throughput economical neutralization assay to investigate the epidemiology of adenovirus type 2 (Ad2)-specific immunity in China and developed countries, including in a Chinese Human immunodeficiency virus (HIV)-1-infected population, and to guide the application of Ad2-vectored vaccines. We established a FluoroSpot-based anti-Ad2-virus neutralization assay using a recombinant replication-deficient Ad2 that expresses enhanced green fluorescent protein and standardized the critical parameters, including the choice of cell line, cell concentration, viral infective dose, and incubation time. The sera of 561 healthy individuals from China and developed countries and from 230 HIV-1-infected Chinese individuals were screened with this assay for Nabs against Ad2. The prevalence of anti-Ad2 NAbs was high in both China (92.2%) and developed countries (86.9%). Of the Ad2-seropositive individuals, 64.6% in China and 77.4% in developed countries had high NAb titers (> 810). The frequency of anti-Ad2 NAbs was higher in Anhui (97.5%) than in Beijing (88.7%). Their prevalence differed significantly according to age in Beijing, but not in Anhui Province, but by sex in neither province. Ad2 seroprevalence was as high among HIV-1-infected individuals (88.7%) as among healthy individuals (92.2%) in China. In conclusion, a simple, intuitive, high-throughput, economical fluorescence-based neutralization assay was developed to determine anti-Ad2 NAbs titers. Ad2 exposure was high in both healthy and HIV-1-infected populations in China, so vectors based on Ad2 may be inappropriate for human vaccines.

Pre-existing neutralizing antibody mitigates B cell dysregulation and enhances the Env-specific antibody response in SHIV-infected rhesus macaques.

Our central hypothesis is that protection against HIV infection will be powerfully influenced by the magnitude and quality of the B cell response. Although sterilizing immunity, mediated by pre-formed abundant and potent antibodies is the ultimate goal for B cell-targeted HIV vaccine strategies, scenarios that fall short of this may still confer beneficial defenses against viremia and disease progression. We evaluated the impact of sub-sterilizing pre-existing neutralizing antibody on the B cell response to SHIV infection. Adult male rhesus macaques received passive transfer of a sub-sterilizing amount of polyclonal neutralizing immunoglobulin (Ig) purified from previously infected animals (SHIVIG) or control Ig prior to intra-rectal challenge with SHIVSF162P4 and extensive longitudinal sampling was performed. SHIVIG treated animals exhibited significantly reduced viral load and increased de novo Env-specific plasma antibody. Dysregulation of the B cell profile was grossly apparent soon after infection in untreated animals; exemplified by a ≈50% decrease in total B cells in the blood evident 2–3 weeks post-infection which was not apparent in SHIVIG treated animals. IgD+CD5+CD21+ B cells phenotypically similar to marginal zone-like B cells were highly sensitive to SHIV infection, becoming significantly decreased as early as 3 days post-infection in control animals, while being maintained in SHIVIG treated animals, and were highly correlated with the induction of Env-specific plasma antibody. These results suggest that B cell dysregulation during the early stages of infection likely contributes to suboptimal Env-specific B cell and antibody responses, and strategies that limit this dysregulation may enhance the host’s ability to eliminate HIV.

Cardiac gene therapy with adeno-associated virus-based vectors.

Cardiac gene therapy with adeno-associated virus (AAV)-based vectors is emerging as an entirely new platform to treat, or even cure, so far intractable cardiac disorders. This review describes our current knowledge of cardiac AAV gene therapy with a particular focus on the biggest obstacle for the successful translation of cardiac AAV gene therapy into the clinic, namely the efficient delivery of the therapeutic gene to the myocardium. We summarize the significant recent progress that has been made in treating heart failure in preclinically relevant animal models with AAV gene therapy and the recent results of clinical trials with cardiac AAV gene therapy for the treatment of heart failure. We also discuss the benefits and shortcomings of the currently available delivery methods of AAV to the heart. Finally, we describe the current state of identifying novel AAV variants that have enhanced tropism for human cardiomyocytes and that show increased resistance to preexisting neutralizing antibodies. Here, we describe the successes and challenges in cardiac AAV gene therapy, a treatment modality that has the potential to transform current treatment approaches for cardiac diseases.

OA13.07 Intrapleural Modified Vaccine Strain Measles Virus Therapy for Patients with Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MM) remains an almost universally fatal disease with limited treatment options. Preclinical models indicate the preferential oncolytic activity of the modified vaccine strain measles virus carrying the gene for the human sodium-iodine symporter (NIS) – MV-NIS. Intraperitoneal and intravenous administration of MV-NIS was recently found to be potentially effective in patients with refractory ovarian cancer and multiple myeloma. However, whether MV-NIS is directly oncolytic or triggers an anti-tumor immune response remains unclear. We conducted a phase I dose escalation study with 3+3 design and ongoing maximal tolerated dose (MTD) expansion cohort. MV-NIS was administered as first or second line therapy via a tunneled intrapleural catheter to patients with MM. MV-NIS dose ranged from 108 TICID50 to 9 x 109 TICID50. In the absence of dose limiting toxicity and disease progression, patients received up to 6 cycles of MV-NIS therapy (Phase I). Currently additional patients are being randomized between a single and multiple cycles. MV-NIS infection and replication are monitored by Iodine123 SPECT/CT (Phase I only) as well as by RT-PCR and/or plaque-assay. Anti-tumor immunity is monitored in the blood and pleural fluid and patients are followed clinically by chest CT using the modified RECIST criteria. Twelve patients (3/dose level) received MV-NIS therapy. There were no dose limiting adverse events and therapy was well tolerated. The best therapeutic response was stable disease, which was achieved at 1 month by 8/12 evaluable patients (67%). Median overall survival was 449 days (95%CI: 221, 484) (∼15 months) (4/12 patients remain alive), and median progression free survival was 63 days (95% CI: 33, 174) (∼2 months). MV infection and replication were detectable by RT-PCR and plaque assay in the pleural fluid between 24-72 hours after treatment. I123 SPECT-CT demonstrated only marginal viral gene expression in a single patient treated with the highest dose level. MV-NIS therapy effectively boosted pre-existing anti-MV neutralizing antibody responses in the plasma and pleural fluid of most patients. We observed a transient inflammatory response in the pleural space after MV-NIS administration. In addition, induction or boosting of anti-tumor antibody responses was observed. The intrapleural administration of MV-NIS is safe, resulted in stable disease for 67% of patients and may be associated with favorable overall survival in MM. While there was only transient infection and viral replication, we observed the induction of anti-tumor immune responses supportive of potential long-term therapeutic impact. The study continues with the MTD expansion cohort.

Oncolytic Adenovirus Complexes Coated with Lipids and Calcium Phosphate for Cancer Gene Therapy.

Oncolytic adenovirus (OncoAd) is a promising therapeutic agent for treating cancer. However, the therapeutic potential of OncoAd is hindered by hepatic sequestration and the host immune response in vivo. Here, we constructed a PEG/Lipids/calcium phosphate (CaP)-OncoAd (PLC-OncoAd) delivery system for ZD55-IL-24, an oncolytic adenovirus that carries the IL-24 gene. The negatively charged PLC-ZD55-IL-24 were disperse and resisted serum-induced aggregation. Compared to naked ZD55-IL-24, the systemic administration of PLC-ZD55-IL-24 in BALB/c mice resulted in reduced liver sequestration and systemic toxicity and evaded the innate immune response. In addition, masking the surface of OncoAd protected it from neutralization by pre-existing neutralizing antibody. PLC-OncoAd achieved efficient targeted delivery in Huh-7-bearing nude mice, and intravenous administration of a high dose of PLC-ZD55-IL-24 increased therapeutic efficacy without inducing toxicity. The developed PLC-OncoAd delivery system represents a promising improvement for oncolytic adenovirus-based cancer gene therapy in vivo.

Cell-Based Measurement of Neutralizing Antibodies Against Adeno-Associated Virus (AAV).

In recent years gene therapy using adeno-associated viral (AAV) vectors to treat cardiac disease has seen an unprecedented surge, owing to its safety, low immunogenicity relative to other vectors and high and long-term transduction efficiency. This field has also been hampered by the presence of preexisting neutralizing antibodies, not only in patients participating in clinical trials but also in preclinical large animal models. These conflicting circumstances have generated the need for a simple, efficient, and fast assay to screen subjects for the presence of neutralizing antibodies, or lack thereof, in order for them to be included in gene therapy trials.

Tumor-specific delivery of biologics by a novel T-cell line HOZOT.

“Cell-in-cell” denotes an invasive phenotype in which one cell actively internalizes in another. The novel human T-cell line HOZOT, established from human umbilical cord blood, was shown to penetrate a variety of human cancer cells but not normal cells. Oncolytic viruses are emerging as biological therapies for human cancers; however, efficient viral delivery is limited by a lack of tumor-specific homing and presence of pre-existing or therapy-induced neutralizing antibodies. Here, we report a new, intriguing approach using HOZOT cells to transmit biologics such as oncolytic viruses into human cancer cells by cell-in-cell invasion. HOZOT cells were successfully loaded via human CD46 antigen with an attenuated adenovirus containing the fiber protein of adenovirus serotype 35 (OBP-401/F35), in which the telomerase promoter regulates viral replication. OBP-401/F35–loaded HOZOT cells were efficiently internalized into human cancer cells and exhibited tumor-specific killing by release of viruses, even in the presence of anti-viral neutralizing antibodies. Moreover, intraperitoneal administration of HOZOT cells loaded with OBP-401/F35 significantly suppressed peritoneally disseminated tumor growth in mice. This unique cell-in-cell property provides a platform for selective delivery of biologics into human cancer cells, which has important implications for the treatment of human cancers.

Intramuscular administration of AAV overcomes pre-existing neutralizing antibodies in rhesus macaques

The seroprevalence of neutralizing antibodies (NAbs) to adeno-associated viral (AAV) vector capsids may preclude a percentage of the population from receiving gene therapy, particularly following systemic vector administration. We hypothesized that the use of intramuscular (IM) administration of AAV vectors might circumvent this issue. IM injections were used to administer AAV8 vectors expressing either secreted or non-secreted transgenes into mice and the influence of NAbs supplied by pre-administration of pooled human IgG on transgene expression was evaluated. We then studied the impact of naturally occurring pre-existing AAV8 NAbs on expression of a secreted transgene following IM vector delivery in rhesus macaques. Finally, we evaluated the ability to readminister AAV vectors via IM injections in rhesus macaques. In mice, the presence of AAV8 NAbs had no effect on gene expression in the injected skeletal muscle. However, liver transgene expression following hepatic distribution of the vector was ablated. In rhesus macaques, naturally occurring pre-existing AAV8 NAb titers of ⩽1:160 had no effect on expression levels of a secreted transgene after IM delivery of the vector. Additionally, readministration of AAV vectors was possible by IM injection into the previously injected muscle groups, with no effect on transgene expression by the original vector. Therefore, the presence of pre-existing NAbs in the human population should not preclude subjects from receiving gene therapy by IM administration of the vector so long as sufficient levels of secreted transgene expression can be produced without the involvement of liver.

High frequency of pre-existing neutralizing antibody responses in patients with dengue during an outbreak in Central Brazil.

BackgroundThis study aims to identify dengue neutralizing antibody response in patients with dengue from a well-characterized cohort during an outbreak in central Brazil, 2012–2013.MethodsWe analyzed paired samples from 40 patients with severe dengue and 20 patients with dengue. Eligibility criteria were: IgM, NS1Ag and/or RT-PCR positivity and positive IgG result. Plaque reduction neutralization test (PRNT50) from DENV-1 to DENV-4 was performed to identify serotype-specific NAbs response. An infecting serotype was defined as ≥4-fold increase in DENV NAbs in paired samples. Monotypic response was classified as PRNT50 ≥ 1/20 to only one DENV serotype, and multitypic response was considered to be PRNT50 ≥ 1/20 to two or more serotypes simultaneously.ResultsPatients were mainly adults. Virological dengue infection was confirmed by RT-PCR: DENV-4(n = 14) and DENV-1(n = 10). Forty-four out of 60(73.3 %) patients had NAbs to DENV-4, DENV-1(68.3 %), DENV-2(68.3 %) and DENV-3(61.6 %) respectively. Fifteen percent of the cases presented monotypic response, whereas 85 % had multitypic response. DENV-4 infected-patients presented the greatest difference in PRNT50 titers compared with other serotypes. Pre-existing DENV NAbs was not correlated with disease severity. This was the first time that DENV-4 was implicated in an epidemic in the region.ConclusionOur data indicates high exposure of multiple DENV serotypes in all age groups in the pre-dengue vaccine era and also previous to Zika virus introduction in Brazil.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1867-6) contains supplementary material, which is available to authorized users.

Sequential and Simultaneous Immunization of Rabbits with HIV-1 Envelope Glycoprotein SOSIP.664 Trimers from Clades A, B and C.

We have investigated the immunogenicity in rabbits of native-like, soluble, recombinant SOSIP.664 trimers based on the env genes of four isolates of human immunodeficiency virus type 1 (HIV-1); specifically BG505 (clade A), B41 (clade B), CZA97 (clade C) and DU422 (clade C). The various trimers were delivered either simultaneously (as a mixture of clade A + B trimers) or sequentially over a 73-week period. Autologous, Tier-2 neutralizing antibody (NAb) responses were generated to the clade A and clade B trimers in the bivalent mixture. When delivered as boosting immunogens to rabbits immunized with the clade A and/or clade B trimers, the clade C trimers also generated autologous Tier-2 NAb responses, the CZA97 trimers doing so more strongly and consistently than the DU422 trimers. The clade C trimers also cross-boosted the pre-existing NAb responses to clade A and B trimers. We observed heterologous Tier-2 NAb responses albeit inconsistently, and with limited overall breath. However, cross-neutralization of the clade A BG505.T332N virus was consistently observed in rabbits immunized only with clade B trimers and then boosted with clade C trimers. The autologous NAbs induced by the BG505, B41 and CZA97 trimers predominantly recognized specific holes in the glycan shields of the cognate virus. The shared location of some of these holes may account for the observed cross-boosting effects and the heterologous neutralization of the BG505.T332N virus. These findings will guide the design of further experiments to determine whether and how multiple Env trimers can together induce more broadly neutralizing antibody responses.

Chimpanzee adenovirus vector-based avian influenza vaccine completely protects mice against lethal challenge of H5N1

Highly pathogenic avian H5N1 viruses may give rise to the next influenza pandemic due to their reassortment and mutation of the genome. Vaccine against this virus is important for coping with its potential threat. Chimpanzee adenovirus (Ad) vectors are a novel type of vaccine vectors that share the advantages of human serotype Ad vectors but without being affected by pre-existing human neutralizing antibody to the vaccine vector. Based on a replication-deficient chimpanzee Ad vector, AdC7, we generated a novel H5N1 vaccine candidate AdC7-H5HA that expresses H5N1 Hemagglutinin(HA). When tested in mice, the vaccine significantly reduced the virus load and pathological lesions in the lung tissues, and conferred complete protection against lethal challenge by a homologous virus. Mechanistically, the AdC7-H5HA vaccine can induce both HA-specific humoral and cell-mediated immune responses in mice. Also, sera transfer experiments demonstrated that neutralizing antibodies alone could provide protection. In conclusion, our results show that chimpanzee Ad vector expressing influenza virus HA may represent a promising vaccine candidate for H5N1 viruses and other influenza virus subtypes.

Bait flavor preference and immunogenicity of ONRAB baits in domestic dogs on the Navajo Nation, Arizona

Abstract Rabies is responsible for an estimated 59,000 human deaths worldwide, and domestic dogs are the primary reservoir and vector of the disease. Among some nations, widespread vaccination has led to elimination of rabies in domestic dogs, yet dogs are still susceptible to rabies infection from interactions with wildlife reservoirs. On Tribal lands in the United States, less than 20% of domestic dogs are vaccinated for rabies, and parenteral vaccination is often unfeasible. Oral rabies vaccination may provide a solution, but a suitable bait flavor and vaccine must be identified. We evaluated 5 bait flavors (bacon, cheese, egg, fish, and sweet) in pairwise flavor-preference trials using placebo Ultralite baits in 26 domestic dogs on the Navajo Nation, Arizona. Each bait flavor was offered a total of 104 times. In all paired comparisons, bacon was more frequently preferred to the alternative. The sweet flavor (the flavor used operationally for oral rabies vaccine (ORV) distribution in Canada) was least preferred. Forty domestic dogs were offered baits containing ONRAB ORV: 14 received the sweet-flavored bait packet and 26 received bacon-flavored baits. Serum was collected from dogs before vaccination and at day 14 and 30 or 37 days after vaccination. Thirty-seven dogs consumed the baits, 2 baits (both sweet flavored) were chewed and spit out, and 1 (sweet flavored) was swallowed without apparent chewing (gulped). Eight dogs had preexisting rabies virus neutralizing antibody (RVNA) titers and 13 naive dogs failed to seroconvert during the study period. Overall, 27 dogs (67.5%) showed increased RVNA titers after vaccination, including 1 dog who chewed and spit out the bait and all dogs with positive baseline RVNA titers. Geometric mean titers for all dogs that seroconverted during the study period peaked at day 14 (1.2 IU/mL; n = 24) and decreased slightly by the final sampling day (0.8 IU/mL; n = 27). We conclude that bacon flavor may be a suitable bait flavor for ORV distribution in loosely kept or free-roaming domestic dogs. Seroconversion among dogs who ingested ONRAB-filled baits was variable. Why 13 dogs who consumed ORV baits failed to seroconvert remains unknown. Additional research to improve seroconversion rates in domestic dogs after vaccination with ONRAB is recommended.

Albumin-binding adenoviruses circumvent pre-existing neutralizing antibodies upon systemic delivery

Recombinant adenoviruses are used as vaccines, gene therapy vectors, and oncolytic viruses. However, the efficacy of such therapies is limited by pre-existing neutralizing antibodies (NAbs), especially when the virus is administered systemically for a wider biodistribution or to reach multiple metastases. To protect adenovirus against NAbs we inserted an albumin-binding domain (ABD) in the main adenovirus capsid protein, the hexon. This domain binds serum albumin to shield the virus upon systemic administration. The ABD-modified adenoviruses bind human and mouse albumin and maintain the infectivity and replication capacity in presence of NAbs. In pre-immunized mice non-modified viruses are completely neutralized, whereas ABD-modified viruses preserve the ability to transduce target organs, induce oncolysis, or generate immune responses to expressed proteins. Our results indicate that albumin coating of the virus capsid represents an effective approach to evade pre-existing NAbs. This strategy has translational relevance in the use of adenovirus for gene therapy, cancer virotherapy, and vaccination.