Virus-like particle-based vaccines targeting the Anopheles mosquito salivary protein, TRIO.

Malaria is a highly lethal infectious disease caused by Plasmodium parasites. These parasites are transmitted to vertebrate hosts when mosquitoes of the Anopheles genus probe for a blood meal. Sporozoites, the infectious stage of Plasmodium , transit to the liver within hours of injection into the dermis. Vaccine efforts are hindered by the complexity of the parasite's lifecycle and the speed at which the infection is established in the liver. In an effort to enhance immunity against Plasmodium , we produced a virus-like particle (VLP)-based vaccine displaying an epitope of TRIO, an Anopheles salivary protein which has been shown to enhance mobility and dispersal of sporozoites in the dermis. Previous work demonstrated that passive immunization with TRIO offered protection from liver infection and acted synergistically with a Plasmodium targeted vaccine. Immunization of mice with TRIO VLPs resulted in high-titer and long-lasting antibody responses that did not significantly drop for over 18 months post-immunization. TRIO VLPs were similarly immunogenic when combined with an anti-malaria vaccine targeting the L9 epitope of the Plasmodium falciparum circumsporozoite protein.However, when used in a malaria challenge mouse model, TRIO VLPs only provided modest protection from infection and did not boost the protection provided by L9 VLPs.

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 Integrated Signaling Threshold Initiates IgG Response toward Virus-like Immunogens.

Class-switched neutralizing Ab (nAb) production is rapidly induced upon many viral infections. However, due to the presence of multiple components in virions, the precise biochemical and biophysical signals from viral infections that initiate nAb responses remain inadequately defined. Using a reductionist system of synthetic virus-like structures, in this study, we show that a foreign protein on a virion-sized liposome can serve as a stand-alone danger signal to initiate class-switched nAb responses without T cell help or TLR but requires CD19. Introduction of internal nucleic acids (iNAs) obviates the need for CD19, lowers the epitope density (ED) required to elicit the Ab response, and transforms these structures into highly potent immunogens that rival conventional virus-like particles in their ability to elicit strong Ag-specific IgG. As early as day 5 after immunization, structures harboring iNAs and decorated with just a few molecules of surface Ag at doses as low as 100 ng induced all IgG subclasses of Ab in mice and reproduced the IgG2a/2c restriction that is long observed in live viral infections. These findings reveal a shared mechanism for the nAb response in mice. High ED is capable but not necessary for driving Ab secretion. Instead, even a few molecules of surface Ag, when combined with nucleic acids within these structures, can trigger strong IgG production. As a result, the signaling threshold for induction of IgG in individual B cells is set by dual signals originating from both ED on the surface and the presence of iNAs within viral particulate immunogens.

Virus-like particle (VLP)-based vaccine targeting tau phosphorylated at Ser396/Ser404 (PHF1) site outperforms phosphorylated S199/S202 (AT8) site in reducing tau pathology and restoring cognitive deficits in the rTg4510 mouse model of tauopathy.

Tauopathies, including Alzheimer's disease (AD) and Frontotemporal Dementia (FTD), are histopathologically defined by the aggregation of hyperphosphorylated pathological tau (pTau) as neurofibrillary tangles in the brain. Site-specific phosphorylation of tau occurs early in the disease process and correlates with progressive cognitive decline, thus serving as targetable pathological epitopes for immunotherapeutic development. Previously, we developed a vaccine (Qß-pT181) displaying phosphorylated Thr181 tau peptides on the surface of a Qß bacteriophage virus-like particle (VLP) that induced robust antibody responses, cleared pathological tau, and rescued memory deficits in a transgenic mouse model of tauopathy. Here we report the characterization and comparison of two additional Qß VLP-based vaccines targeting the dual phosphorylation sites Ser199/Ser202 (Qß-AT8) and Ser396/Ser404 (Qß-PHF1). Both Qß-AT8 and Qß-PHF1 vaccines elicited high-titer antibody responses against their pTau epitopes. However, only Qß-PHF1 rescued cognitive deficits, reduced soluble and insoluble pathological tau, and reactive microgliosis in a 4-month rTg4510 model of FTD. Both sera from Qß-AT8 and Qß-PHF1 vaccinated mice were specifically reactive to tau pathology in human AD post-mortem brain sections. These studies further support the use of VLP-based immunotherapies to target pTau in AD and related tauopathies and provide potential insight into the clinical efficacy of various pTau epitopes in the development of immunotherapeutics.

Minimal Determinants for Lifelong Antiviral Antibody Responses in Mice from a Single Exposure to Virus-like Immunogens at Low Doses.

The durability of an antibody (Ab) response is highly important for antiviral vaccines. However, due to the complex compositions of natural virions, the molecular determinants of Ab durability from viral infection or inactivated viral vaccines have been incompletely understood. Here we used a reductionist system of liposome-based virus-like structures to examine the durability of Abs from primary immune responses in mice. This system allowed us to independently vary fundamental viral attributes and to do so without additional adjuvants to model natural viruses. We show that a single injection of protein antigens (Ags) orderly displayed on a virion-sized liposome is sufficient to induce a long-lived neutralizing Ab (nAb) response. The introduction of internal nucleic acids dramatically modulates the magnitude of Ab responses without an alteration of the long-term kinetic trends. These Abs are characterized by very slow off-rates of ~0.0005 s-1, which emerged as early as day 5 after injection and these off-rates are comparable to that of affinity-matured monoclonal Abs. A single injection of these structures at doses as low as 100 ng led to lifelong nAb production in mice. Thus, a minimal virus-like immunogen can give rise to potent and long-lasting antiviral Abs in a primary response in mice without live infection. This has important implications for understanding both live viral infection and for optimizing vaccine design.

Virus-like particle (VLP)-based vaccine targeting tau phosphorylated at Ser396/Ser404 (PHF1) site outperforms phosphorylated S199/S202 (AT8) site in reducing tau pathology and restoring cognitive deficits in the rTg4510 mouse model of tauopathy.

Tauopathies, including Alzheimer's disease (AD) and Frontotemporal Dementia (FTD), are histopathologically defined by the aggregation of hyperphosphorylated pathological tau (pTau) as neurofibrillary tangles in the brain. Site-specific phosphorylation of tau occurs early in the disease process and correlates with progressive cognitive decline, thus serving as targetable pathological epitopes for immunotherapeutic development. Previously, we developed a vaccine (Qß-pT181) displaying phosphorylated Thr181 tau peptides on the surface of a Qß bacteriophage virus-like particle (VLP) that induced robust antibody responses, cleared pathological tau, and rescued memory deficits in a transgenic mouse model of tauopathy. Here we report the characterization and comparison of two additional Qß VLP-based vaccines targeting the dual phosphorylation sites Ser199/Ser202 (Qß-AT8) and Ser396/Ser404 (Qß-PHF1). Both Qß-AT8 and Qß-PHF1 vaccines elicited high-titer antibody responses against their pTau epitopes. However, only Qß-PHF1 rescued cognitive deficits, reduced soluble and insoluble pathological tau, and reactive microgliosis in a 4-month rTg4510 model of FTD. Both sera from Qß-AT8 and Qß-PHF1 vaccinated mice were specifically reactive to tau pathology in human AD post-mortem brain sections. These studies further support the use of VLP-based immunotherapies to target pTau in AD and related tauopathies and provide potential insight into the clinical efficacy of various pTau epitopes in the development of immunotherapeutics.

An integrated signaling threshold initiates IgG response towards virus-like immunogens.

Class-switched neutralizing antibody (nAb) production is rapidly induced upon many viral infections. However, due to the presence of multiple components in typical virions, the precise biochemical and biophysical signals from viral infections that initiate nAb responses remain inadequately defined. Using a reductionist system of synthetic virus-like structures (SVLS) containing minimal, highly purified biochemical components commonly found in enveloped viruses, here we show that a foreign protein on a virion-sized liposome can serve as a stand-alone danger signal to initiate class-switched nAb responses in the absence of cognate T cell help or Toll-like receptor signaling but requires CD19, the antigen (Ag) coreceptor on B cells. Introduction of internal nucleic acids (iNAs) obviates the need for CD19, lowers the epitope density (ED) required to elicit the Ab response and transforms these structures into highly potent immunogens that rival conventional virus-like particles in their ability to elicit strong Ag-specific IgG. As early as day 5 after immunization, structures harbouring iNAs and decorated with just a few molecules of surface Ag at doses as low as 100 ng induced all IgG subclasses of Ab known in mice and reproduced the IgG2a/2c restriction that has been long observed in live viral infections. These findings reveal a shared mechanism for nAb response upon viral infection. High ED is capable but not necessary for driving Ab secretion in vivo . Instead, even a few molecules of surface Ag, when combined with nucleic acids within these structures, can trigger strong antiviral IgG production. As a result, the signaling threshold for the induction of neutralizing IgG is set by dual signals originating from both ED on the surface and the presence of iNAs within viral particulate immunogens. One-sentence summary: Reconstitution of minimal viral signals necessary to initiate antiviral IgG.

Minimal determinants for lifelong antiviral antibody responses in BALB/c mice from a single exposure to virus-like immunogens at low doses.

However, due to the complex compositions of natural virions, the molecular determinants of Ab durability from viral infection or inactivated viral vaccines have been incompletely understood. Here we used a reductionist system of liposome-based virus-like structures to examine the durability of Abs in primary immune responses in mice. This system allowed us to independently vary fundamental viral attributes and to do so without additional adjuvants to model natural viruses. We show that a single injection of antigens (Ags) orderly displayed on a virion-sized liposome is sufficient to induce a long-lived neutralizing Ab (nAb) response. Introduction of internal nucleic acids dramatically modulates the magnitude of long-term Ab responses without alteration of the long-term kinetic trends. These Abs are characterized by exceptionally slow off-rates of ~0.0005 s-1, which emerged as early as day 5 after injection and these off-rates are comparable to that of affinity-matured monoclonal Abs. A single injection of these structures at doses as low as 100 ng led to lifelong nAb production in BALB/c mice. Thus, a minimal virus-like immunogen can give rise to potent and long-lasting antiviral Abs in a primary response in mice without live infection. This has important implications for understanding both live viral infection and for optimized vaccine design.

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.

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 virus-like particle-based bivalent PCSK9 vaccine lowers LDL-cholesterol levels in non-human primates.

Elevated low-density lipoprotein cholesterol (LDL-C) is an important risk factor in the development of atherosclerotic cardiovascular disease (ASCVD). Inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9), a negative regulator of LDL-C metabolism, have emerged as promising approaches for reducing elevated LDL-C levels. Here, we evaluated the cholesterol-lowering efficacy of virus-like particle (VLP) based vaccines that target epitopes found within the LDL receptor (LDL-R) binding domain of PCSK9. In both mice and non-human primates, a bivalent VLP vaccine targeting two distinct epitopes on PCSK9 elicited strong and durable antibody responses and lowered cholesterol levels. In macaques, a VLP vaccine targeting a single PCSK9 epitope was only effective at lowering LDL-C levels in combination with statins, whereas immunization with the bivalent vaccine lowered LDL-C without requiring statin co-administration. These data highlight the efficacy of an alternative, vaccine-based approach for lowering LDL-C.

A Virus-like particle-based bivalent PCSK9 vaccine lowers LDL-cholesterol levels in Non-Human Primates.

Elevated low-density lipoprotein cholesterol (LDL-C) is an important risk factor in the development of atherosclerotic cardiovascular disease (ASCVD). Inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9), a negative regulator of LDL-C metabolism, have emerged as promising approaches for reducing elevated LDL-C levels. Here, we evaluated the cholesterol lowering efficacy of virus-like particle (VLP) based vaccines that target epitopes found within the LDL receptor (LDL-R) binding domain of PCSK9. In both mice and non-human primates, a bivalent VLP vaccine targeting two distinct epitopes on PCSK9 elicited strong and durable antibody responses and lowered cholesterol levels. In macaques, a VLP vaccine targeting a single PCSK9 epitope was only effective at lowering LDL-C levels in combination with statins, whereas immunization with the bivalent vaccine lowered LDL-C without requiring statin co-administration. These data highlight the efficacy of an alternative, vaccine-based approach for lowering LDL-C.

The Immunogenicity of a VLP-based Malaria Vaccine Targeting CSP in Pregnant and Neonatal Mice.

Maternal antibodies are passively transferred to the fetus via the placenta during gestation and can play an important role in protecting the newborn from infection. For example, in malaria-endemic regions, maternal antibodies likely provide substantial protection against Plasmodium falciparum malaria in the first 6 months of life. However, circulating maternal antibodies can also interfere with vaccine efficacy. Here, we used a mouse maternal transfer model to evaluate whether maternal antibodies interfere with the responsiveness to a virus-like particle (VLP)-based vaccine targeting the CIS43 epitope of the malaria circumsporozoite protein (CSP). We found immunized dams passively transfer to pups high levels of anti-CSP IgG antibodies that steadily decline as the animals age. We also found that the neonatal offspring of immunized mice do not respond to de novo immunization with the CIS43-targeted VLP vaccine until maternal antibody titers decline below an inhibitory threshold. These findings may have important implications for delineating the delicate balance between protection conferred by maternal antibodies and the offspring's ability to respond to immunization.

A method for mapping the linear epitopes targeted by the natural antibody response to Zika virus infection using a VLP platform technology.

Zika virus (ZIKV), a mosquito-borne pathogen, is associated with neurological complications in adults and congenital abnormalities in newborns. There are no vaccines or treatments for ZIKV infection. Understanding the specificity of natural antibody responses to ZIKV could help inform vaccine efforts. Here, we used a technology called Deep Sequence-Coupled Biopanning to map the targets of the human antibody responses to ZIKV infection. A bacteriophage virus-like particle (VLP) library displaying overlapping linear peptides derived from the ZIKV polyprotein was generated. The library was panned using IgG from 23 ZIKV-infected patients from Panama and deep sequencing identified common targets of anti-ZIKV antibodies within the ZIKV envelope glycoprotein. These included epitopes within the fusion loop within domain II and four epitopes within domain III. Additionally, we showed that VLPs displaying selected epitopes elicited antibodies that bound to native ZIKV envelope protein but failed to prevent infection in a mouse challenge model.

A vaccine targeting the L9 epitope of the malaria circumsporozoite protein confers protection from blood-stage infection in a mouse challenge model.

Pre-erythrocytic malaria vaccines that induce high-titer, durable antibody responses can potentially provide protection from infection. Here, we engineered a virus-like particle (VLP)-based vaccine targeting a recently described vulnerable epitope at the N-terminus of the central repeat region of the Plasmodium falciparum circumsporozoite protein that is recognized by the potently inhibitory monoclonal antibody L9 and show that immunization with L9 VLPs induces strong antibody responses that provide protection from blood-stage malaria in a mouse infection model.

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.

Engineering an Antibody V Gene-Selective Vaccine.

The ligand-binding surface of the B cell receptor (BCR) is formed by encoded and non-encoded antigen complementarity determining regions (CDRs). Genetically reproducible or 'public' antibodies can arise when the encoded CDRs play deterministic roles in antigen recognition, notably within human broadly neutralizing antibodies against HIV and influenza virus. We sought to exploit this by engineering virus-like-particle (VLP) vaccines that harbor multivalent affinity against gene-encoded moieties of the BCR antigen binding site. As proof of concept, we deployed a library of RNA bacteriophage VLPs displaying random peptides to identify a multivalent antigen that selectively triggered germline BCRs using the human VH gene IGVH1-2*02. This VLP selectively primed IGHV1-2*02 BCRs that were present within a highly diversified germline antibody repertoire within humanized mice. Our approach thus provides methodology to generate antigens that engage specific BCR configurations of interest, in the absence of structure-based information.

A VLP-based vaccine targeting ANGPTL3 lowers plasma triglycerides in mice.

Elevated triglycerides (TGs) are an important risk factor for the development of coronary heart disease (CHD) and in acute pancreatitis. Angiopoietin-like proteins 3 (ANGPTL3) and 4 (ANGPTL4) are critical regulators of TG metabolism that function by inhibiting the activity of lipoprotein lipase (LPL), which is responsible for hydrolyzing triglycerides in lipoproteins into free fatty acids. Interestingly, individuals with loss-of-function mutations in ANGPTL3 and ANGPTL4 have low plasma TG levels, have a reduced risk of CHD, and are otherwise healthy. Consequently, interventions targeting ANGPTL3 and ANGPTL4 have emerged as promising new approaches for reducing elevated TGs. Here, we developed virus-like particle (VLP) based vaccines that target the LPL binding domains of ANGPTL3 and ANGPTL4. ANGPTL3 VLPs and ANGPTL4 VLPs are highly immunogenic in mice and vaccination with ANGPTL3 VLPs, but not ANGPTL4 VLPs, was associated with reduced steady state levels of TGs. Immunization with ANGPTL3 VLPs rapidly cleared circulating TG levels following an oil gavage challenge and enhanced plasma LPL activity. These data indicate that targeting ANGPTL3 by active vaccination is a potential alternative to other ANGPTL3-inhibiting therapies.

RNA Phage VLP-Based Vaccine Platforms.

Virus-like particles from a variety of RNA bacteriophages have turned out to be useful platforms for delivery of vaccine antigens in a highly immunogenic format. Here we update the current state of development of RNA phage VLPs as platforms for presentation of diverse antigens by genetic, enzymatic, and chemical display methods.