A two-dose regimen of Qß virus-like particle-based vaccines elicit protective antibodies against heroin and fentanyl.

Opioid overdoses and the growing rate of opioid use disorder (OUD) are major public health concerns, particularly in the United States. Current treatment approaches for OUD have failed to slow the growth of the opioid crisis. Opioid vaccines have shown pre-clinical success in targeting multiple different opioid drugs. However, the need for many immunizations can limit their clinical implementation. In this study, we investigate the development of novel opioid vaccines by independently targeting fentanyl and the active metabolites of heroin using a bacteriophage virus-like particle (VLP) vaccine platform. We establish the successful conjugation of haptens to bacteriophage Qß VLPs and demonstrate immunogenicity of Qß-fentanyl, Qß-morphine, and Qß-6-acetylmorphine in animal models after one or two immunizations. We show that in independently or in combination, these vaccines elicit high-titer, high-avidity, and durable antibody responses. Moreover, we reveal their protective capacities against heroin or fentanyl challenge after two immunizations. Overall, these findings establish Qß-VLP conjugated vaccines for heroin and fentanyl as very promising opioid vaccine candidates.

An Approach for Antigen-Agnostic Identification of Virus-Like Particle-Displayed Epitopes that Engage Specific Antibody V Gene Regions.

Antibody complementarity determining regions (CDRs) participate in antigen recognition, but not all participate equally in antigen binding. Here we describe a technique for discovering strong, specific binding partners to defined motifs within the CDRs of chimeric, engineered antibodies using affinity selection and counter-selection of epitopes displayed on bacteriophage MS2-based virus-like particles (VLPs). As an example, we show how this technique can be used to identify families of VLPs that interact with antibodies displaying the CDRs encoded by the germline precursor of a broadly neutralizing monoclonal antibody against HIV-1.

A bacteriophage virus-like particle vaccine against oxycodone elicits high-titer and long-lasting antibodies that sequester drug in the blood.

Opioid use disorder (OUD) and opioid overdoses are public health emergencies. In 2021, 80,000 opioid overdose associated deaths were reported in the United States. Despite the availability of treatment strategies, including medications for opioid use disorder (MOUD) and naloxone, opioid overdoses continue to increase at an alarming rate. Opioid vaccines are a novel approach to combat the growing crisis with several candidates recently entering human clinical trials. In this study, we investigated Qß bacteriophage virus-like particles (VLPs) as a vaccine platform for immunogenic display of oxycodone. A derivative of oxycodone was conjugated to pre-formed Qß VLPs using a sulfhydryl-amine reactive heterobifunctional crosslinker with high loading of oxycodone. In mice, intramuscular immunization with Qß-oxycodone elicited high-titer, high-avidity and long-lasting antibody responses. Qß-oxycodone was also immunogenic after storage at ambient room temperature for over two weeks, demonstrating that the vaccine is highly thermostable. In mice, immunization with Qß-oxycodone elicited antibodies that sequester oxycodone in the serum, an important mechanism for preventing the adverse effects of opioid activity. Finally, Qß-oxycodone is immunogenic in nonhuman primates, eliciting serum oxycodone antibodies after intramuscular immunization of rhesus macaques. These data establish Qß-oxycodone as a promising opioid vaccine candidate.

Streamlined Data Analysis Pipeline for Deep Sequence-Coupled Biopanning Identification of Pathogen-Specific Antibody Responses in Serum.

Deep sequence-coupled biopanning (DSCB) is a powerful tool that couples affinity selection of a bacteriophage MS2 virus-like particle peptide display platform with deep sequencing. While this approach has been used successfully to investigate pathogen-specific antibody responses in human sera, data analysis is time-consuming and complicated. Here, we describe a streamlined data analysis method for DSCB using MATLAB, expanding the potential for this approach to be deployed rapidly and consistently.

Unbiased Identification of Dengue Virus Non-Structural Protein 1 Peptides for Use in Vaccine Design.

Dengue virus (DENV) is a global health problem, with over half of the world's population at risk for infection. Despite this, there is only one licensed vaccine available to prevent infection and safety concerns limit immunization to only a subset of individuals. Most dengue virus vaccine efforts attempt to evoke broadly neutralizing antibodies against structural proteins. However, eliciting antibodies to block the activity of viral proteins involved in pathogenesis could be a useful complementary approach. Studies suggest that non-structural protein 1, which participates in disruption of the endothelial barrier and is hypothesized to play a significant role in the progression to severe dengue, could be a promising target for vaccine efforts. Here, we used an unbiased approach to identify peptide epitopes of dengue virus non-structural protein 1 that could evoke antibodies that bind to NS1 from all 4 serotypes and also bind to DENV-infected cells. DENV-2 NS1 peptides were generated such that 35 overlapping 15 amino acid peptides represented the entire NS1 protein. These peptides were each chemically conjugated to bacteriophage virus-like particles (VLP) and used to immunize mice. Sera were then screened for IgG to cognate peptide as well as binding to recombinant hexameric NS1 from all four DENV serotypes as well as binding to DENV-2 infected cells by microscopy. From these data, we identified several peptides that were able to elicit antibodies that could bind to infected cells as well as DENV NS1. These peptides and their homologues in the corresponding NS1 of other DENV serotypes could be used as potential immunogens to elicit binding antibodies to NS1. Future studies will investigate the functional and protective capacities of antibodies elicited by these immunogens against DENV NS1.

Epitope-Based Vaccines against the Chlamydia trachomatis Major Outer Membrane Protein Variable Domain 4 Elicit Protection in Mice.

Chlamydia trachomatis (Ct) is the most common bacterial sexual transmitted pathogen, yet a vaccine is not currently available. Here, we used the immunogenic bacteriophage MS2 virus-like particle (VLP) technology to engineer vaccines against the Ct major outer membrane protein variable domain 4 (MOMP-VD4), which contains a conserved neutralizing epitope (TTLNPTIAG). A previously described monoclonal antibody to the MOMP-VD4 (E4 mAb) is capable of neutralizing all urogenital Ct serovars and binds this core epitope, as well as several non-contiguous amino acids. This suggests that this core epitope may require conformational context in order to elicit neutralizing antibodies to Ct. In order to identify immunogens that could elicit neutralizing antibodies to the TTLNPTIAG epitope, we used two approaches. First, we used affinity selection with a bacteriophage MS2-VLP library displaying random peptides in a constrained, surface-exposed loop to identify potential E4 mAb mimotopes. After four rounds of affinity selection, we identified a VLP-displayed peptide (HMVGSTKWTN) that could bind to the E4 mAb and elicited serum IgG that bound weakly to Ct elementary bodies by ELISA. Second, two versions of the core conserved TTLNPTIAG epitope (TTLNPTIAG and TTLNPTIAGA) were recombinantly expressed on the coat protein of the MS2 VLP in a constrained, surface-exposed loop. Mouse immune sera IgG bound to Ct elementary bodies by ELISA. Immunization with these MS2 VLPs provided protection from vaginal Chlamydia infection in a murine challenge model. These data suggest that short peptide epitopes targeting the MOMP-VD4 could be appropriate for Ct vaccine design when displayed on an immunogenic bacteriophage VLP vaccine platform.

Immunogenicity and Protective Capacity of a Virus-like Particle Vaccine against Chlamydia trachomatis Type 3 Secretion System Tip Protein, CT584.

An effective vaccine against Chlamydia trachomatis is urgently needed as infection rates continue to rise and C. trachomatis causes reproductive morbidity. An obligate intracellular pathogen, C. trachomatis employs a type 3 secretion system (T3SS) for host cell entry. The tip of the injectosome is composed of the protein CT584, which represents a potential target for neutralization with vaccine-induced antibody. Here, we investigate the immunogenicity and efficacy of a vaccine made of CT584 epitopes coupled to a bacteriophage virus-like particle (VLP), a novel platform for Chlamydia vaccines modeled on the success of HPV vaccines. Female mice were immunized intramuscularly, challenged transcervically with C. trachomatis, and assessed for systemic and local antibody responses and bacterial burden in the upper genital tract. Immunization resulted in a 3-log increase in epitope-specific IgG in serum and uterine homogenates and in the detection of epitope-specific IgG in uterine lavage at low levels. By contrast, sera from women infected with C. trachomatis and virgin controls had similarly low titers to CT584 epitopes, suggesting these epitopes are not systemically immunogenic during natural infection but can be rendered immunogenic by the VLP platform. C. trachomatis burden in the upper genital tract of mice varied after active immunization, yet passive protection was achieved when immune sera were pre-incubated with C. trachomatis prior to inoculation into the genital tract. These data demonstrate the potential for antibody against the T3SS to contribute to protection against C. trachomatis and the value of VLPs as a novel platform for C. trachomatis vaccines.

Expansion and Refinement of Deep Sequence-Coupled Biopanning Technology for Epitope-Specific Antibody Responses in Human Serum.

Identifying the specific epitopes targeted by antibodies elicited in response to infectious diseases is important for developing vaccines and diagnostics. However, techniques for broadly exploring the specificity of antibodies in a rapid manner are lacking, limiting our ability to quickly respond to emerging viruses. We previously reported a technology that couples deep sequencing technology with a bacteriophage MS2 virus-like particle (VLP) peptide display platform for identifying pathogen-specific antibody responses. Here, we describe refinements that expand the number of patient samples that can be processed at one time, increasing the utility of this technology for rapidly responding to emerging infectious diseases. We used dengue virus (DENV) as a model system since much is already known about the antibody response. Sera from primary DENV-infected patients (n = 28) were used to pan an MS2 bacteriophage VLP library displaying all possible 10-amino-acid peptides from the DENV polypeptide. Selected VLPs were identified by deep sequencing and further investigated by enzyme-linked immunosorbent assay. We identified previously described immunodominant regions of envelope and nonstructural protein-1, as well as a number of other epitopes. Our refinement of the deep sequence-coupled biopanning technology expands the utility of this approach for rapidly investigating the specificity of antibody responses to infectious diseases.

Antibodies to Variable Domain 4 Linear Epitopes of the Chlamydia trachomatis Major Outer Membrane Protein Are Not Associated with Chlamydia Resolution or Reinfection in Women.

Chlamydia trachomatis is an obligate intracellular bacterium. C. trachomatis infection is the most prevalent bacterial sexually transmitted infection and can lead to pelvic inflammatory disease and infertility in women. There is no licensed vaccine for C. trachomatis prevention, in part due to gaps in our knowledge of C. trachomatis-specific immune responses elicited during human infections. Previous investigations of the antibody response to C. trachomatis have identified immunodominant antigens and antibodies that can neutralize infection in cell culture. However, epitope-specific responses to C. trachomatis are not well characterized, and the impact of these antibodies on infection outcome is unknown. We recently developed a technology called deep sequence-coupled biopanning that uses bacteriophage virus-like particles to display peptides from antigens and affinity select against human serum IgG. Here, we used this technology to map C. trachomatis-specific antibodies in groups of women with defined outcomes following C. trachomatis infection: (i) C. trachomatis negative upon presentation for treatment ("spontaneous resolvers"), (ii) C. trachomatis negative at a 3-month follow-up visit after treatment ("nonreinfected"), and (iii) C. trachomatis positive at a 3-month follow-up after treatment ("reinfected"). This analysis yielded immunodominant epitopes that had been previously described but also identified new epitopes targeted by human antibody responses to C. trachomatis We focused on human antibody responses to the C. trachomatis variable domain 4 serovar-conserved region of the major outer membrane protein (VD4-MOMP), a previously described immunodominant epitope. All three groups of women produced IgG to the VD4-MOMP, suggesting that detection of serum antibodies to VD4-MOMP in women with urogenital C. trachomatis infection is not associated with protection against reinfection.IMPORTANCEC. trachomatis infection is the most common bacterial sexually transmitted infection, and infection in women can lead to pelvic inflammatory disease and infertility. No licensed vaccine exists to prevent C. trachomatis infection, and investigations of the natural immune response may inform the design of targeted vaccines for C. trachomatis Our study fills a gap in knowledge regarding the epitope specificity of antibody responses that are elicited in response to C. trachomatis infection in women. We identified several new B cell epitopes for C. trachomatis antigens and confirmed B cell epitopes that have been identified by other methods. Our finding that women produce antibodies to the VD4-MOMP regardless of infection outcome provides insight into vaccine development, suggesting that vaccines targeting VD4-MOMP may need to elicit higher-titer antibody responses than natural infection imparts or that additional vaccine targets should be pursued in the future.

Assessing Antibody Specificity in Human Serum Using Deep Sequence-Coupled Biopanning.

Affinity selection using phage-display technologies is a powerful tool for identifying the peptide epitopes of monoclonal antibodies. Coupling affinity selection with deep sequencing technologies allows for the broad assessment of selectant populations. Here, we describe a method for using a phage-display platform to assess antibody specificity in human serum. We describe the method with reference to the bacteriophage MS2 virus-like particle (VLP) platform, but it can be adapted to other phage-display technologies as well.

Sidestream smoke exposure increases the susceptibility of airway epithelia to adenoviral infection.

BACKGROUND: Although significant epidemiological evidence indicates that cigarette smoke exposure increases the incidence and severity of viral infection, the molecular mechanisms behind the increased susceptibility of the respiratory tract to viral pathogens are unclear. Adenoviruses are non-enveloped DNA viruses and important causative agents of acute respiratory disease. The Coxsackievirus and adenovirus receptor (CAR) is the primary receptor for many adenoviruses. We hypothesized that cigarette smoke exposure increases epithelial susceptibility to adenovirus infection by increasing the abundance of apical CAR. METHODOLOGY AND FINDINGS: Cultured human airway epithelial cells (CaLu-3) were used as a model to investigate the effect of sidestream cigarette smoke (SSS), mainstream cigarette smoke (MSS), or control air exposure on the susceptibility of polarized respiratory epithelia to adenoviral infection. Using a Cultex air-liquid interface exposure system, we have discovered novel differences in epithelial susceptibility between SSS and MSS exposures. SSS exposure upregulates an eight-exon isoform of CAR and increases adenoviral entry from the apical surface whilst MSS exposure is similar to control air exposure. Additionally, the level of cellular glycogen synthase kinase 3ß (GSK3ß) is downregulated by SSS exposure and treatment with a specific GSK3ß inhibitor recapitulates the effects of SSS exposure on CAR expression and viral infection. CONCLUSIONS: This is the first time that SSS exposure has been shown to directly enhance the susceptibility of a polarized epithelium to infection by a common respiratory viral pathogen. This work provides a novel understanding of the impact of SSS on the burden of respiratory viral infections and may lead to new strategies to alter viral infections. Moreover, since GSK3ß inhibitors are under intense clinical investigation as therapeutics for a diverse range of diseases, studies such as these might provide insight to extend the use of clinically relevant therapeutics and increase the understanding of potential side effects.