Intrastructural Help: Harnessing T Helper Cells Induced by Licensed Vaccines for Improvement of HIV Env Antibody Responses to Virus-Like Particle Vaccines.

Induction of persistent antibody responses by vaccination is generally thought to depend on efficient help by T follicular helper cells. Since the T helper cell response to HIV Env may not be optimal, we explored the possibility of improving the HIV Env antibody response to virus-like particle (VLP) vaccines by recruiting T helper cells induced by commonly used licensed vaccines to provide help for Env-specific B cells. B cells specific for the surface protein of a VLP can internalize the entire VLP and thus present peptides derived from the surface and core proteins on their major histocompatibility complex class II (MHC-II) molecules. This allows T helper cells specific for the core protein to provide intrastructural help for B cells recognizing the surface protein. Consistently, priming mice with an adjuvanted Gag protein vaccine enhanced the HIV Env antibody response to subsequent booster immunizations with HIV VLPs. To harness T helper cells induced by the licensed Tetanolpur vaccines, HIV VLPs that contained T helper cell epitopes of tetanus toxoid were generated. Tetanol-immunized mice raised stronger antibody responses to immunizations with VLPs containing tetanus toxoid T helper cell epitopes but not to VLPs lacking these epitopes. Depending on the priming immunization, the IgG subtype response to HIV Env after the VLP immunization could also be modified. Thus, harnessing T helper cells induced by other vaccines appears to be a promising approach to improve the HIV Env antibody response to VLP vaccines.IMPORTANCE Induction of HIV Env antibodies at sufficient levels with optimal Fc effector functions for durable protection remains a challenge. Efficient T cell help may be essential to induce such a desirable antibody response. Here, we provide proof of concept that T helper cells induced by a licensed vaccine can be harnessed to provide help for HIV Env-specific B cells and to modulate the Env-specific IgG subtype response.

Generation of T follicular helper cells in vitro: requirement for B-cell receptor cross-linking and cognate B- and T-cell interaction.

The minimum requirements for in vitro modelling of natural CD4+ T-cell differentiation into T follicular helper (Tfh) cells are still under investigation. We co-cultured wild-type and T-cell receptor (TCR) transgenic CD4+ T cells from naive mice with dendritic cells and B-cell receptor (BCR) transgenic B cells in the presence of HIV-derived virus-like particles containing matched B-cell and T-cell epitopes. This co-culturing induced co-expression of Tfh-master regulator transcription factor BCL-6 and CXCR5 in up to 10% of the wild-type and up to 40% of the TCR-transgenic CD4+ T cells. Phenotypic markers, production of interleukin-21 and isotype switching of the B cells to IgG1 further indicated a helper function of the induced Tfh cells in vitro. Dendritic cells supported the generation of functional Tfh cells, but were unable to induce them without cognate B cells. Hence, our study presents a robust experimental system for efficient generation of functionally active Tfh cells in vitro and confirms the importance of cognate B- and T-cell cross-talk for the Tfh differentiation process.

Enhancing the Quality of Antibodies to HIV-1 Envelope by GagPol-Specific Th Cells.

The importance of Fc-dependent effector functions of Abs induced by vaccination is increasingly recognized. However, vaccination of mice against HIV envelope (Env) induced a skewed Th cell response leading to Env-specific Abs with reduced effector function. To overcome this bias, GagPol-specific Th cells were harnessed to provide intrastructural help for Env-specific B cells after immunization with virus-like particles containing GagPol and Env. This led to a balanced Env-specific humoral immune response with a more inflammatory Fc glycan profile. The increased quality in the Ab response against Env was confirmed by FcγR activation assays. Because the Env-specific Th cell response was also biased in human vaccinees, intrastructural help is an attractive novel approach to increase the efficacy of prophylactic HIV Env-based vaccines and may also be applicable to other particulate vaccines.

Nanoparticle-based B-cell targeting vaccines: Tailoring of humoral immune responses by functionalization with different TLR-ligands.

Induction of an appropriate type of humoral immune response during vaccination is essential for protection against viral and bacterial infections. We recently observed that biodegradable calcium phosphate (CaP) nanoparticles coated with proteins efficiently targeted and activated naïve antigen-specific B-cells in vitro. We now compared different administration routes for CaP-nanoparticles and demonstrated that intramuscular immunization with such CaP-nanoparticles induced stronger immune responses than immunization with monovalent antigen. Additional functionalization of the CaP-nanoparticles with TRL-ligands allowed modulating the IgG subtype response and the level of mucosal IgA antibodies. CpG-containing CaP-nanoparticles were as immunogenic as a virus-like particle vaccine. Functionalization of CaP-nanoparticles with T-helper cell epitopes or CpG also allowed overcoming lack of T-cell help. Thus, our results indicate that CaP-nanoparticle-based B-cell targeting vaccines functionalized with TLR-ligands can serve as a versatile platform for efficient induction and modulation of humoral immune responses in vivo.

Application of the PAMONO-Sensor for Quantification of Microvesicles and Determination of Nano-Particle Size Distribution.

The PAMONO-sensor (plasmon assisted microscopy of nano-objects) demonstrated an ability to detect and quantify individual viruses and virus-like particles. However, another group of biological vesicles-microvesicles (100-1000 nm)-also attracts growing interest as biomarkers of different pathologies and needs development of novel techniques for characterization. This work shows the applicability of a PAMONO-sensor for selective detection of microvesicles in aquatic samples. The sensor permits comparison of relative concentrations of microvesicles between samples. We also study a possibility of repeated use of a sensor chip after elution of the microvesicle capturing layer. Moreover, we improve the detection features of the PAMONO-sensor. The detection process utilizes novel machine learning techniques on the sensor image data to estimate particle size distributions of nano-particles in polydisperse samples. Altogether, our findings expand analytical features and the application field of the PAMONO-sensor. They can also serve for a maturation of diagnostic tools based on the PAMONO-sensor platform.

DNA vaccines encoding DEC205-targeted antigens: immunity or tolerance?

Targeting of antigens to the endocytic uptake receptor DEC205 resulted in enhanced antigen presentation by dendritic cells (DCs). In combination with adjuvants for DC maturation, proteins coupled to an antibody against DEC205 induced strong pathogen-specific immune responses, whereas without additional adjuvant tolerance could be induced. As less is known about DNA vaccines encoding DEC205-targeted antigens, we explored the immunogenicity and efficacy of a dendritic cell-targeted DNA vaccine against influenza A virus (IAV) delivered by electroporation. Although coupling of haemagglutinin to a single-chain antibody against DEC205 enhanced antigen presentation on MHC class II and activation of T-cell receptor-transgenic CD4 T cells, the T-cell responses induced by the targeted DNA vaccine in wild-type BALB/c mice were significantly reduced compared with DNA encoding non-targeted antigens. Consistently, these mice were less protected against an IAV infection. Adoptive transfer experiments were performed to assess the fate of the antigen-specific T cells in animals vaccinated with DNA encoding DEC205-targeted antigens. By this, we could exclude the general deletion of antigen-specific T cells as cause for the reduced efficacy, but observed a local expansion of antigen-specific regulatory T cells, which could suppress the activation of effector cells. In conclusion, DNA vaccines encoding DEC205-targeted antigens induce peripheral tolerance rather than immunity in our study. Finally, we evaluated our DNA vaccines as prophylactic or therapeutic treatment in an allergen-induced asthma mouse model.

Application of surface plasmon resonance imaging technique for the detection of single spherical biological submicrometer particles.

Recent proof-of-principle studies demonstrated the suitability of the surface plasmon resonance imaging (SPRi) technique for the detection of individual submicrometer and nanoparticles in solutions. In the current study, we used the SPRi technique for visualization of the binding of round-shaped viruses (inactivated influenza A virus) and virus-like particles (human immunodeficiency virus (HIV)-based virus-like particles) to the functionalized sensor surface. We show the applicability of the SPRi technique for the detection of individual virus-like particles in buffers without serum as well as in buffers containing different concentrations of serum. Furthermore, we prove the specificity of visualized binding events using two different pseudotypes of HIV virus-like particles. We also demonstrate the applicability of the SPRi technique for the determination of relative particle concentrations in solutions. Moreover, we suggest a technical approach, which allows enhancing the magnitude of binding signals. Our studies indicate that the SPRi technique represents an efficient research tool for quantification and characterization of biological submicrometer objects such as viruses or virus-like particles, for example.

Targeting the antigen encoded by adenoviral vectors to the DEC205 receptor modulates the cellular and humoral immune response.

Replication-defective adenoviral vectors have emerged as promising vaccine candidates for diseases relying on strong CD8(+) T-cell responses for protection. In this study, we modified a non-replicative adenoviral vector to selectively deliver, in situ, an encoded ovalbumin (OVA) model antigen to dendritic cells (DCs). Efficient uptake and presentation of OVA was achieved through fusion of the antigen to a single-chain antibody directed against DEC205, an endocytic receptor expressed on DCs. The immunogenicity of the vaccine was thereby enhanced as demonstrated by elevated antibody levels and increased T-cell responses after low-dose vaccination with 10(7) viral particles compared with a non-targeted control. Nevertheless, after immunization with higher doses of the targeted vaccine, the capacity of vaccine-induced CD8(+) T cells to produce the cytokine IL-2 was diminished and the CD8(+) T-cell response was dominated by an effector memory phenotype (CD62L(-)/CD127(+)) in contrast to the effector phenotype (CD62L(-)/CD127(-)) observed after non-targeted antigen delivery. Interestingly, the protective capacity of the non-targeted vaccine was superior to that of the targeted vaccine in an antigen-specific vaccinia virus infection as well as in a tumor challenge model. In the latter, the low dose of the DC-targeted vaccine also conferred partial protection from tumor growth, demonstrating dose-dependent effects of the DC-targeting on the quality of the vaccine-induced immune response. Significant differences could be observed in regard to the antibody pattern, the functional and phenotypic T-cell repertoire, and to the protective capacity.

Oriented display of HIV-1 Env trimers by a novel coupling strategy enhances B cell activation and phagocytosis.

Introduction: Conformationally stabilized Env trimers have been developed as antigens for the induction of neutralizing antibodies against HIV-1. However, the non-glycosylated immunodominant base of these soluble antigens may compete with the neutralizing antibody response. This has prompted attempts to couple Env trimers to organic or inorganic nanoparticles with the base facing towards the carrier. Such a site-directed coupling could not only occlude the base of the trimer, but also enhance B cell activation by repetitive display. Methods: To explore the effect of an ordered display of HIV-1 Env on microspheres on the activation of Env-specific B cells we used Bind&Bite, a novel covalent coupling approach for conformationally sensitive antigens based on heterodimeric coiled-coil peptides. By engineering a trimeric HIV-1 Env protein with a basic 21-aa peptide (Peptide K) extension at the C-terminus, we were able to covalently biotinylate the antigen in a site-directed fashion using an acidic complementary peptide (Peptide E) bearing a reactive site and a biotin molecule. This allowed us to load our antigen onto streptavidin beads in an oriented manner. Results: Microspheres coated with HIV-1 Env through our Bind&Bite system showed i) enhanced binding by conformational anti-HIV Env broadly neutralizing antibodies (bNAbs), ii) reduced binding activity by antibodies directed towards the base of Env, iii) higher Env-specific B cell activation, and iv) were taken-up more efficiently after opsonization compared to beads presenting HIV-1 Env in an undirected orientation. Discussion: In comparison to site-directed biotinylation via the Avi-tag, Bind&Bite, offers greater flexibility with regard to alternative covalent protein modifications, allowing selective modification of multiple proteins via orthogonal coiled-coil peptide pairs. Thus, the Bind&Bite coupling approach via peptide K and peptide E described in this study offers a valuable tool for nanoparticle vaccine design where surface conjugation of correctly folded antigens is required.

Influence of AAV vector tropism on long-term expression and Fc-γ receptor binding of an antibody targeting SARS-CoV-2.

Long-acting passive immunization strategies are needed to protect immunosuppressed vulnerable groups from infectious diseases. To further explore this concept for COVID-19, we constructed Adeno-associated viral (AAV) vectors encoding the human variable regions of the SARS-CoV-2 neutralizing antibody, TRES6, fused to murine constant regions. An optimized vector construct was packaged in hepatotropic (AAV8) or myotropic (AAVMYO) AAV capsids and injected intravenously into syngeneic TRIANNI-mice. The highest TRES6 serum concentrations (511 µg/ml) were detected 24 weeks after injection of the myotropic vector particles and mean TRES6 serum concentrations remained above 100 µg/ml for at least one year. Anti-drug antibodies or TRES6-specific T cells were not detectable. After injection of the AAV8 particles, vector mRNA was detected in the liver, while the AAVMYO particles led to high vector mRNA levels in the heart and skeletal muscle. The analysis of the Fc-glycosylation pattern of the TRES6 serum antibodies revealed critical differences between the capsids that coincided with different binding activities to murine Fc-γ-receptors. Concomitantly, the vector-based immune prophylaxis led to protection against SARS-CoV-2 infection in K18-hACE2 mice. High and long-lasting expression levels, absence of anti-drug antibodies and favourable Fc-γ-receptor binding activities warrant further exploration of myotropic AAV vector-based delivery of antibodies and other biologicals.

GagPol-specific CD4⁺ T-cells increase the antibody response to Env by intrastructural help.

BACKGROUND: Immunization of rhesus macaques against Gag of SIV resulted in a more rapid appearance of Env antibodies after infection with SIV or SHIV challenge viruses although the vaccines lacked an Env component. We therefore explored whether T helper cells specific for internal HIV proteins could provide intrastructural help for Env-specific B cells and thus increase the Env antibody response. RESULTS: Mice were immunized by adenoviral vector or DNA vaccines against GagPol and then boosted with virus-like particles (VLP) containing GagPol and Env. Env-specific antibody levels after the VLP booster immunizations were significantly higher in GagPol-immunized mice than in mock-vaccinated controls. Adoptive transfer of CD4+ T cells from GagPol-immunized mice also enhanced the Env antibody response to VLP immunization in the recipient mice. Depending on the presence of VLPs, co-cultivation of CD4+ T cells from GagPol-primed mice with BCR transgenic B cells specific for a protein presented on the surface of the VLPs also resulted in the activation of the B and T cells. CONCLUSIONS: Our study indicates that GagPol-specific T helper cells may provide intrastructural help for Env antibody responses. This cross-talk between immune responses directed against different components of the retroviral particle may be relevant for the immunopathogenesis of retroviral infections and allow to improve virus like particle vaccine approaches against HIV.

Risk of immunodeficiency virus infection may increase with vaccine-induced immune response.

To explore the efficacy of novel complementary prime-boost immunization regimens in a nonhuman primate model for HIV infection, rhesus monkeys primed by different DNA vaccines were boosted with virus-like particles (VLP) and then challenged by repeated low-dose rectal exposure to simian immunodeficiency virus (SIV). Characteristic of the cellular immune response after the VLP booster immunization were high numbers of SIV-specific, gamma interferon-secreting cells after stimulation with inactivated SIV particles, but not SIV peptides, and the absence of detectable levels of CD8(+) T cell responses. Antibodies specific to SIV Gag and SIV Env could be induced in all animals, but, consistent with a poor neutralizing activity at the time of challenge, vaccinated monkeys were not protected from acquisition of infection and did not control viremia. Surprisingly, vaccinees with high numbers of SIV-specific, gamma interferon-secreting cells were infected fastest during the repeated low-dose exposures and the numbers of these immune cells in vaccinated macaques correlated with susceptibility to infection. Thus, in the absence of protective antibodies or cytotoxic T cell responses, vaccine-induced immune responses may increase the susceptibility to acquisition of immunodeficiency virus infection. The results are consistent with the hypothesis that virus-specific T helper cells mediate this detrimental effect and contribute to the inefficacy of past HIV vaccination attempts (e.g., STEP study).

Immunogenicity of Recombinant Lipid-Based Nanoparticle Vaccines: Danger Signal vs. Helping Hand.

Infectious diseases are a predominant problem in human health. While the incidence of many pathogenic infections is controlled by vaccines, some pathogens still pose a challenging task for vaccine researchers. In order to face these challenges, the field of vaccine development has changed tremendously over the last few years. For non-replicating recombinant antigens, novel vaccine delivery systems that attempt to increase the immunogenicity by mimicking structural properties of pathogens are already approved for clinical applications. Lipid-based nanoparticles (LbNPs) of different natures are vesicles made of lipid layers with aqueous cavities, which may carry antigens and other biomolecules either displayed on the surface or encapsulated in the cavity. However, the efficacy profile of recombinant LbNP vaccines is not as high as that of live-attenuated ones. This review gives a compendious picture of two approaches that affect the immunogenicity of recombinant LbNP vaccines: (i) the incorporation of immunostimulatory agents and (ii) the utilization of pre-existing or promiscuous cellular immunity, which might be beneficial for the development of tailored prophylactic and therapeutic LbNP vaccine candidates.

Antigen-dependent modulation of immune responses to antigen-Fc fusion proteins by Fc-effector functions.

Background: Fc-fusion proteins have been successfully developed for therapeutic purposes, but are also a promising platform for the fast generation and purification of immunogens capable of inducing strong humoral immune responses in preclinical immunization studies. As the Fc-portion of immunoglobulins fused to an antigen confers functional properties of the parental antibody, such as dimerization, binding to Fc-receptors and complement activation, several studies reported that Fc-fusion proteins elicit stronger antigen-specific antibody responses than the unfused antigen. However, dimerization or half-life extension of an antigen have also been described to enhance immunogenicity. Methods: To explore the role of Fc-effector functions for the immunogenicity of fusions proteins of viral glycoproteins and Fc fragments, the HIV-1 gp120 and the RBD of SARS-CoV-2 were fused to the wild type muIgG2a Fc fragment or mutants with impaired (LALA-PG) or improved (GASDIE) Fc-effector functions. Results: Immunization of BALB/c mice with DNA vaccines encoding gp120 - Fc LALA-PG induced significantly higher antigen-specific antibody responses than gp120 - Fc WT and GASDIE. In contrast, immunization with DNA vaccines encoding the RBD fused to the same Fc mutants, resulted in comparable anti-RBD antibody levels and similar neutralization activity against several SARS-CoV-2 variants. Conclusion: Depending on the antigen, Fc-effector functions either do not modulate or suppress the immunogenicity of DNA vaccines encoding Fc-antigen fusion proteins.

Modulation of immune responses to liposomal vaccines by intrastructural help.

The encapsulation of HIV-unrelated T helper peptides into liposomal vaccines presenting trimers of the HIV-1 envelope glycoprotein (Env) on the surface (T helper liposomes) may recruit heterologous T cells to provide help for Env-specific B cells. This mechanism called intrastructural help can modulate the HIV-specific humoral immune response. In this study, we used cationic T helper liposomes to induce intrastructural help effects in a small animal model. The liposomes were functionalized with Env trimers by a tag-free approach designed to enable a simplified GMP production. The pre-fusion conformation of the conjugated Env trimers was verified by immunogold electron microscopy (EM) imaging and flow cytometry. The liposomes induced strong activation of Env-specific B cells in vitro. In comparison to previously established anionic liposomes, cationic T helper liposomes were superior in CD4+ T cell activation after uptake by dendritic cells. Moreover, the T helper liposomes were able to target Env-specific B cells in secondary lymphoid organs after intramuscular injection. We also observed efficient T helper cell activation and proliferation in co-cultures with Env-specific B cells in the presence of cationic T helper liposomes. Mouse immunization experiments with cationic T helper liposomes further revealed a modulation of the Env-specific IgG subtype distribution and enhancement of the longevity of antibody responses by ovalbumin- and Hepatitis B (HBV)-specific T cell help. Thus, clinical evaluation of the concept of intrastructural help seems warranted.

T cell independent secondary antibody responses to the envelope protein of simian immunodeficiency virus.

BACKGROUND: During human (HIV) and simian (SIV) immunodeficiency virus infection, loss of CD4+ T cells and progression to AIDS are associated with a decline in antibody titers to the viral Gag protein, while antibodies to the Env protein remain high, suggesting a T cell independent antibody response to Env. RESULTS: To explore differential regulation of Gag and Env antibody responses, immunocompetent BALB/c and T cell deficient nude mice were immunized with virus like particles (VLP) of simian immunodeficiency virus or adenoviral vectors expressing SIV Gag and Env. High levels of antibodies against Gag and Env could only be induced in immunocompetent mice, but not in the immunodeficient mice. Thus, neither cells expressing Env after adenoviral gene transfer nor VLPs induce a T cell independent primary anti-Env antibody response. However, secondary B cell responses to Env, but not to Gag, were observed in immunodeficient mice after transfer of primed B cells and boosting with VLPs or adenoviral vectors expressing Gag and Env. This T cell independent secondary antibody response to Env was reduced after stimulation with VLPs modified to contain monomeric membrane bound gp130 surface subunit of Env and undetectable after injection of soluble gp130. CONCLUSIONS: Membrane-bound trimeric Env seems to be responsible for the maintenance of high levels of anti-Env antibodies during progression to AIDS. This T cell independent secondary antibody response may prevent T cell-dependent affinity maturation and thus contribute to viral immune escape by favoring persistence of non-protective antibodies.

Inhibitors of Deubiquitinating Enzymes Interfere with the SARS-CoV-2 Papain-like Protease and Block Virus Replication In Vitro.

The ubiquitin proteasome system (UPS), particularly its deubiquitinating enzymes (DUBs), play a key role in the replication cycle of coronaviruses. The SARS-CoV-2 papain-like protease (Plpro) is known to process the viral polyproteins to form the replicase transcriptase complex and to counteract the host viral response. Recently, it was shown that this viral protease can also act as a deubiquitinating enzyme. In this study, we demonstrate that certain DUB-Inhibitors (DIs) interfere with SARS-CoV-2 replication. The DIs PR-619 and HBX41108 restrict SARS-CoV-2 in both Vero B4 and human Calu-3 lung cells where cells were infected with a Multiplicity of Infection (MOI) of 0.02. An in vitro protease assay using recombinant Plpro and Amido-4-methylcoumarin (AMC)-conjugated substrate revealed that PR-619 and HBX41108 are able to block the protease at concentrations where the interventions restricted virus replication. In contrast, DIs that do not inhibit Plpro had no influence on virus replication, which indicated that the protease might be at least one major target. Future vertical studies that would gain more insights into the mechanisms of how DUBs effect the replication of SARS-CoV-2 will further validate them as a potential therapeutic target.

Design and Functional Characterization of HIV-1 Envelope Protein-Coupled T Helper Liposomes.

Functionalization of experimental HIV-1 virus-like particle vaccines with heterologous T helper epitopes (T helper VLPs) can modulate the humoral immune response via intrastructural help (ISH). Current advances in the conjugation of native-like HIV-1 envelope trimers (Env) onto liposomes and encapsulation of peptide epitopes into these nanoparticles renders this GMP-scalable liposomal platform a feasible alternative to VLP-based vaccines. In this study, we designed and analyzed customizable Env-conjugated T helper liposomes. First, we passively encapsulated T helper peptides into a well-characterized liposome formulation displaying a dense array of Env trimers on the surface. We confirmed the closed pre-fusion state of the coupled Env trimers by immunogold staining with conformation-specific antibodies. These peptide-loaded Env-liposome conjugates efficiently activated Env-specific B cells, which further induced proliferation of CD4+ T cells by presentation of liposome-derived peptides on MHC-II molecules. The peptide encapsulation process was then quantitatively improved by an electrostatically driven approach using an overall anionic lipid formulation. We demonstrated that peptides delivered by liposomes were presented by DCs in secondary lymphoid organs after intramuscular immunization of mice. UFO (uncleaved prefusion optimized) Env trimers were covalently coupled to peptide-loaded anionic liposomes by His-tag/NTA(Ni) interactions and EDC/Sulfo-NHS crosslinking. EM imaging revealed a moderately dense array of well-folded Env trimers on the liposomal surface. The conformation was verified by liposomal surface FACS. Furthermore, anionic Env-coupled T helper liposomes effectively induced Env-specific B cell activation and proliferation in a comparable range to T helper VLPs. Taken together, we demonstrated that T helper VLPs can be substituted with customizable and GMP-scalable liposomal nanoparticles as a perspective for future preclinical and clinical HIV vaccine applications. The functional nanoparticle characterization assays shown in this study can be applied to other systems of synthetic nanoparticles delivering antigens derived from various pathogens.

Decorated and Encapsulated: Virus-Like Particles Against Viral Infections.

Despite great progress in the field of vaccine development, outbreaks of emerging pathogens and insufficient immunogenicity of some licensed vaccines call for the development of novel technologies in rational vaccine design [...].