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Abstract— Most vaccines work by inducing neutralizing antibodies that target the viral envelope. Enveloped RNA viruses have evolved mechanisms for surface glycoproteins to evade host immune responses, which exhibit substantial variability, even among different strains. Natural infection and vaccines using native forms of surface proteins may induce broadly neutralizing antibodies, yet with low and ineffective levels. Class I membrane-fusion proteins of enveloped RNA viruses, HIV-1, influenza A virus, SARS-CoV-2, yield a stable conformation (so-called “pre-fusion”) in providing fusion between viral and host cell membranes. Modified viral surface proteins that are based on these features induce neutralizing antibodies with activity available against a broad spectrum of circulating strains and make it possible to overcome the difficulties associated with escape/variability of viral antigen.

Purpose: to develop a method for experimental study of the distribution of radionuclide 241Am in human organs and tissues during wound admission to clarify the process of formation of doses of alpha radiation. Material and methods: in clinical practice of Burnazyan FMBC of the FMBA of Russia To improve the method of determination, an experiment was performed to determine the depth of radionuclide on pigskin with the location of 241Am point sources behind different thicknesses. Results: the used methods of measurement, tested on pigskin, allowed to obtain the dependence of the localization depth of radionuclide 241Am on the measured on the surface of the tissue ratios of photons with different energies. Conclusion: Set the ratio of photons with different energies on the thickness of the barrier (depth), and proven methodology allow you to go directly to the planning of experimental studies on the barrier effect created in the bone material, and including a radionuclide, the formation of doses of alpha radiation on the bone marrow.

Previous studies suggest that short peptides from the heptad repeat 2 (HR2) domain of gp41 expressed on the cell surface are more potent inhibitors of HIV-1 entry than soluble analogs. However, their therapeutic potential has only been examined using lentiviral vectors. Here, we aimed to develop CRISPR/Cas9-based fusion inhibitory peptide knock-in (KI) technology for the generation and selection of HIV-1-resistant T cells. First, we embedded a series of HIV-1 fusion inhibitory peptides in CD52, the shortest glycosylphosphatidylinositol (GPI)-anchored protein, which efficiently delivers epitope tags to the cell surface and maintains a sufficient level of KI. Among the seven peptides tested, MT-C34, HP-23L, and 2P23 exhibited significant activity against both cell-free and cell-to-cell HIV-1 infection. The shed variant of MT-C34 provided insufficient protection against HIV-1 due to its low concentration in the culture medium. Using Cas9 plasmids or ribonucleoprotein electroporation and peptide-specific antibodies, we sorted CEM/R5 cells with biallelic KI of MT-C34 and 2P23 peptides at the CXCR4 locus. In combination, these peptides provided a higher level of protection than individual KI. By extending homology arms and cloning donor DNA into a plasmid containing signals for nuclear localization, we achieved KI of MT-C34 into the CXCR4 locus and HIV-1 proviral DNA at levels of up to 35% in the T-cell line and up to 4 to 5% in primary CD4 lymphocytes. Compared to lentiviral delivery, KI resulted in the higher MT-C34 surface expression and stronger protection of lymphocytes from HIV-1. Thus, we demonstrate that KI is a viable strategy for peptide-based therapy of HIV infection.

Previous studies suggest that short peptides from the heptad repeat 2 (HR2) domain of gp41 expressed on the cell surface are more potent inhibitors of HIV-1 entry than soluble analogs. However, their therapeutic potential has only been examined using lentiviral vectors. Here, we aimed to develop CRISPR/Cas9-based fusion inhibitory peptide knock-in (KI) technology for the generation and selection of HIV-1-resistant T cells. First, we cloned a series of HIV-1 fusion inhibitory peptides and embedded them in CD52, the shortest GPI-anchored protein, which efficiently delivers epitope tags to the cell surface and maintains a sufficient level of KI. Among the seven peptides tested, MT-C34, HP-23L, and 2P23 exhibited significant activity against both cell-free and cell-to-cell HIV-1 infection. Unlike membrane-bound peptides, the shed variant of MT-C34 provided insufficient protection against HIV-1 due to its low concentrations in the culture medium. Using Cas9 plasmids or ribonucleoprotein electroporation and cell sorting with antibodies raised against gp41 peptides, we generated CEM/R5 cells with biallelic KI of MT-C34 (embedded in CD52 for expression in lipid rafts) and 2P23 (N-terminally fused to CXCR4). In combination, these peptides provided a higher level of protection than individual KI. By extending homology arms and substituting PCR donor DNA with a plasmid containing signals for nuclear localization, we achieved KI of MT-C34 into CXCR4 loci and HIV-1 proviral DNA at levels of up to 35% in CEM/R5 cells and increased KI occurrence from undetectable to 4-5% in CD4 lymphocytes. Comparative analysis of lentiviral and HDR-based delivery strategies showed that KI led to the higher MT-C34 expression and stronger protection of primary CD4 lymphocytes from HIV-1 than lentiviral transduction, albeit the efficiency of KI needs further improvements in order to meet clinical requirements. Thus, the developed CRISPR/Cas9 platform offers a new opportunity for antiviral peptide delivery with a concomitant precise genetic modification of targeted locus that can be employed to strengthen cell protection against HIV.AUTHOR SUMMARYHIV is a human lentivirus that infects CD4-positive immune cells and, when left untreated, manifests in the fatal disease known as acquired immunodeficiency syndrome. Antiretroviral therapy (ART) is not leading to viral clearance, and HIV persists in the organism as a latent provirus. One way to control infection is to increase the population of HIV-resistant CD4 lymphocytes via entry molecule knockout or expression of different antiviral genes. Peptides from the heptad repeat (HR) domain of gp41 are potent inhibitors of HIV-1 fusion, especially when designed to express on the cell surface. Individual gp41 peptides encoded by therapeutic lentiviral vectors have been evaluated and some have entered clinical trials. However, a CRISPR/Cas9-based gp41 peptide delivery platform that operates through concomitant target gene modification has not yet been developed due to low knock-in (KI) rates in primary cells. Here, we systematically evaluated the antiviral activity of different HR2-peptides cloned into the shortest carrier molecule, CD52. The resulting small-size transgene constructs encoding selected peptides, in combination with improvements to enhance donor vector nuclear import, helped to overcome precise editing restrictions in CD4 lymphocytes. Using KI into CXCR4, we demonstrated different options for target gene modification, effectively protecting edited cells against HIV-1.

A study of the induction of broadly neutralizing antibodies (bNAbs) in HIV-infected patients and vaccinated subjects revealed the main criteria for the formation of bNAbs (the duration of exposure to a viral antigen, the effect of the diversity of HIV variants, and the removal of barriers associated with the Env-dependent defense mechanisms of HIV-1). Modified trimers of the HIV-1 envelope protein (Env) exposed on virus-like particles (VLP) have unique properties: they (i) modulate the exposure of binding sites (bs) of the CD4 receptor and co-receptor; (ii) create steric restrictions for contact with bNAbs; (iii) increase the Env presentation density, thus enhancing the immune response; (iv) form stable trimers that do not induce off-target immune responses; and (v) allow additional modifications to their structure and construction of a platform with immunostimulating molecules. Immunization using a heterologous subtype-cross prime-boost regime with modified trimeric Env is capable of inducing somatic hypermutation levels necessary for the formation of bNAbs.

We defined the effects of the cytoplasmic domain (CT) of the Env glycoprotein on co-receptor usage of HIV-1 by reciprocal exchanges of regions containing V3-V5 loops between CD4-dependent and CD4-independent isolates. Primary HIV-1 isolate Env clones CD8 CXCR4-tropic 92UG046 CT84 with an 84-aa truncated CT domain, CD4 CXCR4-tropic 92UG046, and CD4 CCR5-tropic SF162 with full-length (FL) CT domains were used for comparison. The parental 92UG046 Env with CT84 was not fusogenic, but a chimeric SF162 V3-V5-CT84 with an 84-aa truncated CT domain, which demonstrated a switched co-receptor specificity, exhibited syncytium-formation activity with 3T3T4X4 cells. The wild-type (WT) SF162 Env with CT84 or full-length CT was fusogenic in 3T3T4R5 cells. By exchange of V3-V5 loops, we were able to alter WT SF162 to switch its co-receptor preference, which was not dependent on CT domain length. These results provide evidence that CT domains can induce conformational changes in functional regions of gp120 and determine receptor tropism but do not modulate HIV-1 co-receptor specificity.

An ideal protective HIV-1 vaccine can elicit broadly neutralizing antibodies, capable of preventing HIV transmission. The strategies of designing vaccines include generation of soluble recombinant proteins which mimic the native Env complex and are able to enhance the immunogenicity of gp120. Recent data indicate that the cytoplasmic tail (CT) of the Env protein has multiple functions, which can affect the early steps of infection, as well as viral assembly and antigenic properties. Modifications in the CT can be used to induce conformational changes in functional regions of gp120 and to stabilize the trimeric structure, avoiding immune misdirection and induction of non-neutralizing antibody responses. Env-trimers with modified CTs in virus-like particles (VLPs) are able to induce antibodies with broad spectrum neutralizing activity and high avidity and have the potential for developing an effective vaccine against HIV.

The rapid worldwide spread of human immunodeficiency virus (HIV) mandates the development of successful vaccination strategies. Since live attenuated HIV is not accepted as a vaccine due to safety concerns, virus-like particles (VLPs) offer an attractive safe alternative because they lack the viral genome yet they are perceived by the immune system as a virus particle. We hypothesized that adding immunostimulatory signals to VLPs would enhance their efficacy. To accomplish this we generated chimeric simian immunodeficiency virus (SIV) VLPs containing either glycosylphosphatidylinositol (GPI)-anchored granulocyte-macrophage colony-stimulating factor (GM-CSF) or CD40 ligand (CD40L) and investigated their biological activity and ability to enhance immune responses in vivo. Immunization of mice with chimeric SIV VLPs containing GM-CSF induced SIV Env-specific antibodies as well as neutralizing activity at significantly higher levels than those induced by standard SIV VLPs, SIV VLPs containing CD40L, or standard VLPs mixed with soluble GM-CSF. In addition, mice immunized with chimeric SIV VLPs containing either GM-CSF or CD40L showed significantly increased CD4+- and CD8+-T-cell responses to SIV Env, compared to standard SIV VLPs. Taken together, these results demonstrate that the incorporation of immunostimulatory molecules enhances humoral and cellular immune responses. We propose that anchoring immunostimulatory molecules into SIV VLPs can be a promising approach to augmenting the efficacy of VLP antigens.

The human immunodeficiency virus type 1 (HIV-1) 92UG046 Env protein, obtained from a CD4-independent HIV-1 primary isolate (Zerhouni et al., 2004), has the ability to initiate an infection in HeLa cells expressing CD4 when carrying the full-length (FL) Env, but uses CD8 molecules for receptor-mediated entry when carrying a truncated Env (CT84). To determine whether a specific length or structure in the cytoplasmic tail (CT) is responsible for this alteration of tropism, we compared a series of Env constructs with different CT truncations and the presence or absence of an amphipathic alpha- helical sequence. We found that truncated constructs containing the alpha-helical LLP-2 structure in their CT domains conferred a switch from CD4 to CD8 tropism. The results support the conclusion that the structure of the CT domain can play an important role in determining receptor specificity.

An ideal protective HIV-1 vaccine can elicit broadly neutralizing antibodies, capable of preventing HIV transmission. The strategies of designing vaccines include generation of soluble recombinant proteins which mimic the native Env complex and are able to enhance the immunogenicity of gp120. Recent data indicate that the cytoplasmic tail (CT) of the Env protein has multiple functions, which can affect the early steps of infection, as well as viral assembly and antigenic properties. Modifications in the CT can be used to induce conformational changes in functional regions of gp120 and to stabilize the trimeric structure, avoiding immune misdirection and induction of non-neutralizing antibody responses. Env-trimers with modified CTs in virus-like particles (VLPs) are able to induce antibodies with broad spectrum neutralizing activity and high avidity and have the potential for developing an effective vaccine against HIV.

Sulfonated 5,10,15,20-tetra(1-naphthyl)porphyrin (T1NapS) and 5,10,15,20-tetra(2-naphthyl)porphyrin (T2NapS) and their copper and iron chelates show activity against the human immunodeficiency virus (HIV-1). The porphyrins were prepared by sulfonation of the parent structures with sulfuric acid. More highly sulfonated structures were prepared by sulfonation for longer times. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry showed species with as many as eight sulfonates. Some of the mass spectral peaks for the copper chelates were consistent with loss of water, apparently from intramolecular sulfone formation between two adjacent naphthalene rings that took place during copper insertion. The compounds could be separated using capillary electrophoresis; addition of beta- or gamma-cyclodextrin gave substantially better separation of the components. Activity against HIV was evaluated using an epithelial HeLa-CD4-CCR5 cell line; EC50 values for HIV-1 IIIB and HIV-1 JR-FL ranged from 1 to 15 microg/ml. The compounds exhibit low toxicity for human epithelial cells and have potential as microbicides which might be used to provide protection against sexual transmission of HIV.

We investigated the effect of the conserved tyrosine-based endocytosis motif (YXXΦ) in the cytoplasmic domain of the human immunodeficiency viruses (HIV) envelope protein (Env) on its immunogenicity. Genes with codons optimized for mammalian expression were synthesized for the HIV 89.6 Env with a truncated cytoplasmic domain and a mutant Env in which the tyrosine residue in the YXXΦ motif was changed into a serine. Mutation of the Tyr residue enhanced surface expression of the Env protein. Analysis of immune responses induced by DNA immunization of mice showed that the DNA construct for the Tyr mutant Env induced moderately higher levels of T cell responses. More interestingly, the DNA construct for the mutant Env induced significantly higher levels of antibody responses against the Env protein in comparison to the construct for the wild type Env. Our results suggest that the YXXΦ motif in the HIV Env cytoplasmic domain may play a role in virus evasion of host immune responses through affecting its immunogenicity.

In an attempt to develop an immunization strategy to induce mucosal and circulatory antibodies against SIV antigens, we have investigated the potential of attenuated recombinant vaccinia virus to deliver SIV antigens (gp160 of SIVmac239) to mucosal surfaces of mice. After systemic or mucosal (intragastric, intranasal, or intrarectal) immunization with vaccinia virus-SIV Env recombinants the immune responses against the envelope glycoprotein of SIV, as well as against vaccinia virus antigens, were assessed by ELISA of serum, saliva, and intestinal and vaginal secretions. All immunization routes induced specific antibody titers against gp160 in both serum and external secretions. Recall responses against SIV were found to be acquired after administration of SIVmac239 Env and Gag antigens in a virus-like particle (VLP) form by the same mucosal routes as those used for the priming with recombinant vaccinia virus. The results obtained demonstrate the potential of vaccinia virus recombinants to elicit a primary immune response at mucosal surfaces, which could be enhanced by delivering the same antigen in the form of VLPs.

Two rubella virus DNA vaccines were constructed from a cDNA clone of the rubella virus genomic RNA, one which contained the coding sequences for all three virion proteins (C, E2 and E1) and one which contained the two envelope glycoproteins (E2 and E1). When used to immunize mice via gene gun delivery, both constructs induced an antibody response of equivalent titer to that induced by rubella virus that persisted for at least seven months. A booster injection given four weeks after the initial injection increased antibody titers by over thirty-fold. The antibody response in DNA vaccine-injected mice was directed primarily against the E1 glycoprotein, as was the case in rubella virus-injected mice, and neutralizing activity was detected. These DNA vaccines are thus prototypes for a nonreplicating rubella virus vaccine that could be used in specialized circumstances.

We have compared the immunogenicity of the full-length (FL) SIV envelope (Env) protein and a truncated (T) form of the Env protein which has a short cytoplasmic tail. The Env(T) protein was previously shown to be more fusogenic than Env(FL), has a higher level of incorporation into virus-like particles (VLPs) and membrane vesicles, and expands the viral host range. We have found that mice immunized with VLPs which contained an equal amount of Env(FL) or Env(T) produced similar titres of neutralizing antibody. Comparison of immune responses between animals that received DNA vaccines encoding Env(T) vs. Env (FL) by epidermal delivery demonstrated that a higher level of specific antibody was elicited by Env(T) than Env(FL). This result correlated with a higher level of expression of pCMVEnv(T) than pCMVEnv(FL) observed in vitro. DNA immunization combined with VLP boosting elicited a similar level of neutralizing antibody with both forms of Env proteins. These data indicate that the immunogenicity of Env(FL) and Env(T) is similar, and that either form of Env protein appears to be potentially suitable for use in further development of vaccine preparations.

We investigated the effects of introducing specific sequences that are predicted to affect trimer stability into the CT domain of the SIV Env protein. Two constructs, 3HBai and 3HBaa, with additional GCN4-related sequences in the CT domain (45 aa) had enhanced infectivity, and differed in their fusion activity and trimer stability. Another construct, 3HBii, exhibited a very stable trimeric structure. Pseudotyped virions containing 3HBii retained infectivity despite the lack of syncytia formation. In contrast, 3HBai and 3HBaa, which caused extensive syncytia formation, had a less stable trimeric structure. We observed an inverse correlation between trimer stability and fusion activity but no correlation between syncytia formation activity and infectivity. Quantitative cell–cell fusion assays, analysis of Env incorporation, measurement of ectodomain conformation by CD4 binding, and CCR5 blocking assays indicated differential effects on fusion activity and infectivity of the viruses with Env CT modifications. Differences in interaction with CD4 were not affected by trimer stability and were not related to fusion activity or infectivity. The results indicate that changes in the stability of the CT domain can have significant effects on functional activities of the Env external domain and can impact viral biological properties.

Previously, a modified HIV Env protein with a heterologous membrane anchor was found to be incorporated into HIV virus-like particles (VLPs) at 10-fold-higher levels than those of unmodified Env. To further improve the immunogenicity of such VLPs, membrane-anchored forms of bacterial flagellin (FliC) or a flagellin with a truncated variable region (tFliC) were constructed to be incorporated into the VLPs as adjuvants. HIV-specific immune responses induced by the resulting VLPs were determined in a guinea pig model. The VLPs induce enhanced systemic antibody responses by either systemic or mucosal vaccination and enhanced mucosal immunity by a mucosal immunization route, as demonstrated by high levels of HIV-specific serum IgG and mucosal IgG and IgA. The quality of the antibody responses was also improved, as shown by enhanced neutralization capacity. VLPs incorporating FliC were more effective in inducing systemic responses, while VLPs containing tFliC were more effective in inducing mucosal IgA responses. The IgG titers in sera were found to last for at least 5 months without a significant drop. These results indicate that HIV VLPs incorporating high levels of Env and a molecular adjuvant have excellent potential for further development as a prophylactic HIV vaccine., IMPORTANCE A prophylactic vaccine is urgently needed to control the spread of HIV/AIDS. Antigens inducing strong systemic and mucosal immune responses are promising as vaccines for this mucosally transmitted disease. We found that novel HIV virus-like particles (VLPs) presenting a high level of Env in its native membrane-bound form and coincorporating an innate immune-signaling adjuvant in the same particles were effective in inducing enhanced systemic and mucosal immunity. As new HIV vaccine candidates, these VLPs bridge the gaps of the innate and adaptive, as well as systemic and mucosal, immune responses, providing a new approach for HIV vaccine development.

Momentum spectra of protons emitted at angles of 0.103, 0.139 and 0.157 rad in fragmentation of 9 GeV/ c deuterons on 1 H, 2 H and 12 C nuclei have been measured at the JINR synchrophasotron by using a magnetic spectrometer. It is found that the shape of the high-momentum parts of the spectra is defined only by the deuteron structure and the mechanism of deuteron-nucleon interaction and does not depend on a target. The contributions to the differential cross section of the 1 H(d, p)X reaction have been determined, which are conditioned by elastic and inelastic scatterings of deuteron nucleons on a proton, stripping, their interference, and also the excitation of the pion degrees of freedom. In the framework of the light front dynamics, calculations of the proton spectra with the deuteron structure functions for the Paris, Reid soft core, and Bonn potentials have been performed; a better agreement with the experiment is achieved with the deuteron wave function for the Paris potential. A conclusion is made that the nucleons in a deuteron keep their individuality up to relative motion momenta of ∼ 1 GeV/ c .

CV-1 cells were infected with two recombinant vaccinia viruses carrying the gag gene with deletion of 231 bp from 3' terminus (strain vC5) and env gene (strain vE234L) of human immunodeficiency virus type 1 (HIV-1). Both recombinant proteins synthesized in the cells (p50gag and gp160/120env) were localized predominantly in cell membranes; however, some amount of p50 was found in cell nuclei. Thin-section immunoelectron microscopy showed accumulation of viruslike particles undistinguished from immature HIV-1 virions in the culture medium of the cells infected with vC5. The similar particles containing gag and env proteins were produced into the culture medium when the cells were coinfected with vC5 and vE234L strains. The particles contained heterogeneous cellular RNA, but no virus-specific RNA as shown by Northern blot hybridization. Immunization of the rabbits with purified viruslike particles produced virus-specific antibodies against gag and env proteins. The titer of antibodies was significantly higher than after immunization with cell lysate or recombinant proteins purified from the infected cells. Highly immunogenic HIV-1-like particles containing gag and env proteins but no virus-specific RNA are good candidates for potential vaccine.

Extensive glycosylation and variable loops of the HIV envelope protein (Env) are reported to shield some neutralizing epitopes. Here, we investigated the immunogenicity of mutated HIV Envs presented in virus-like particles (VLPs). We immunized mice with simian human immunodeficiency virus (SHIV) VLPs containing mutant HIV Env with reduced glycosylation (3G), variable loop-deleted mutations (dV1V2), or combinations of both types of mutations (3G-dV2-1G), and evaluated immune responses. Immune sera from mice that received VLPs with modified HIV Envs (3G or dV1V2) showed higher neutralizing activities against the homologous HIV 89.6 virus as well as heterologous viruses when compared with wild type SHIV VLP-immunized mice. Lymphocytes from immunized mice produced HIV Env-specific cytokines, with the 3G-dV2-1G mutant producing high levels of cytokines. Interestingly, both dendritic cells and B cells were found to interact with VLPs suggesting that VLPs are effective immunogens. Therefore, this study suggests that VLPs containing modified HIV Env have the potential to be developed as candidate vaccines capable of inducing cellular and humoral immune responses including neutralizing activities.

Sulfonated porphyrins and phthalocyanines have been shown to have anti-HIV activity and are under consideration as microbicides. Both categories of compounds are small negatively charged molecules and both were previously shown to inhibit cell fusion induced by the HIV Env protein and to block binding of gp120 to the CD4 receptor. In the present study we show that these compounds inhibit transmission of cell-associated HIV, inactivate a broad range of HIV-1 primary isolates and are active against DS polyanion-resistant virus. The compounds tested are active over a range of pH values, and possess no detectable activity against normal bacterial flora. These results support the conclusion that anionic tetrapyrroles are promising candidates as microbicides for HIV prevention.