Triple Combinations of AAV9-Vectors Encoding Anti-HIV bNAbs Provide Long-Term In Vivo Expression of Human IgG Effectively Neutralizing Pseudoviruses from HIV-1 Global Panel.

Anti-human immunodeficiency virus (HIV) broadly neutralizing antibodies (bNAbs) offer a promising approach for the treatment of HIV-1. The current paradigm for antibody therapy involves passive antibody transfer, requiring regular delivery of bNAbs in treating chronic diseases such as HIV-1. An alternative strategy is to use AAV-mediated gene transfer to enable in vivo production of desirable anti-HIV-1 antibodies. In this study, we investigated two sets of triple combinations of AAV9-vectors encoding different bNAbs: N6, 10E8, 10-1074 (CombiMab1), and VRC07-523, PGDM1400, 10-1074 (CombiMab2). We used CBAxC57Bl and C57BL/6 mouse models to characterize rAAV-induced antibody expression and to evaluate the neutralization capacity of mouse sera against a global panel of HIV-1 viral strains. rAAV9-mediated IgG expression varied between bNAb clones and mouse strains, with C57BL/6 mice exhibiting higher bNAb titers following rAAV delivery. Although CombiMab2 treatment elicited a higher IgG titer than CombiMab1, both combinations resulted in neutralization of all the viral strains from the global HIV-1 panel. Our data highlight the potential of AAV vectors as a long-term option for HIV-1 therapy.

Development of MVA-d34 Tetravalent Dengue Vaccine: Design and Immunogenicity.

Dengue fever, an infectious disease that affects more than 100 million people every year, is a global health problem. Vaccination may be the most effective prevention strategy for the disease. However, the development of vaccines against dengue fever is complicated by the high risk of developing an antibody-dependent increase in infection. This article describes the development of an MVA-d34 vaccine against the dengue virus based on a safe and effective MVA viral vector. The DIII domains of the envelope protein (E) of the dengue virus are used as vaccine antigens, as antibodies against these domains do not cause an enhancement of infection. The use of the DIII domains of each of the four dengue virus serotypes made it possible to generate a humoral response against all four dengue virus serotypes in immunized mice. We also showed that the sera of vaccinated mice present virus-neutralizing activity against dengue serotype 2. Thus, the developed MVA-d34 vaccine is a promising candidate vaccine against dengue fever.

Comparative Evaluation of the Activity of Various Lentiviral Vectors Containing Three Anti-HIV Genes.

A promising direction in the treatment of HIV infection is a gene therapy approach based on the insertion of antiviral genes aimed at inhibiting HIV replication into the genome of host cells. We obtained six constructs of lentiviral vectors with different arrangements of three antiviral genes: microRNAs against the CCR5 gene, the gene encoding the C-peptide, and the gene encoding the modified human TRIM5a protein. We found that despite containing the same genes, these vectors were produced at different titers and had different effects on cell viability, transduction efficiency, and expression stability. Comparative evaluation of the antiviral activity of three of the six developed vectors that showed stable expression was carried out using the continuous SupT1 lymphocytic cell line. All of the vectors protected cells from HIV infection: the viral load was several orders of magnitude lower than in control cells, and with one vector, complete cessation of virus growth in modified cells was achieved.

Comparison of Recombinant MVA Selection Methods Based on F13L, D4R and K1L Genes.

Modified vaccinia Ankara (MVA) is a promising vaccine vector due to its highly attenuated phenotype and good immunogenicity. However, obtaining a new recombinant MVA remains a tedious and laborious procedure involving many rounds of plaque purification. Recombinant MVA generation can be greatly improved and facilitated by different selection techniques. Here, we describe a comparison between techniques based on K1L, F13L and D4R genes.

Development of a Universal Epitope-Based Influenza Vaccine and Evaluation of Its Effectiveness in Mice.

Vaccination is an effective and economically viable means of protection against the influenza virus, but due to rapid viral evolution, modern seasonal vaccines are not effective enough. Next-generation vaccines are designed to provide protection against a wide range of influenza virus strains, including pandemic variants. In our work, we made an epitope-based universal vaccine, rMVA-k1-k2, against the influenza virus based on the modified vaccinia Ankara (MVA) vector and using our own algorithms to select epitopes from conserved fragments of the NP, M1 and HA proteins of influenza A and B. We show that double immunization protects mice with a 67% or greater efficiency against viral influenza pneumonia when infected with various strains of the H1N1, H2N2, H3N2 and H5N1 subtypes of influenza A. In animals, the level of protection provided by the rMVA-k1-k2 vaccine was comparable to that provided by the universal M001 and MVA-NP+M1 (Invictus) vaccines, which have shown success in clinical trials, against strains of the H1N1 and H3N2 subtypes.

Development of Modified Vaccinia Virus Ankara-Based Vaccines: Advantages and Applications.

Modified vaccinia virus Ankara (MVA) is a promising viral vector for vaccine development. MVA is well studied and has been widely used for vaccination against smallpox in Germany. This review describes the history of the origin of the virus and its properties as a vaccine, including a high safety profile. In recent years, MVA has found its place as a vector for the creation of vaccines against various diseases. To date, a large number of vaccine candidates based on the MVA vector have already been developed, many of which have been tested in preclinical and clinical studies. We discuss data on the immunogenicity and efficacy of some of these vaccines.

Double and Triple Combinations of Broadly Neutralizing Antibodies Provide Efficient Neutralization of All HIV-1 Strains from the Global Panel.

The use of broadly neutralizing antibodies (bNAbs) is a promising approach to HIV-1 treatment. In this work, we evaluate the neutralizing activity of the following HIV-1 bNAbs: VCR07-523, N6, PGDM1400, CAP256-VRC26.25, 10-1074, PGT128, 10E8, and DH511.11P, which are directed to different Env surface epitopes. We used the global panel of HIV-1 pseudoviruses to analyze the bNAbs' potency and chose the most potent ones. To achieve maximum neutralization breadth and minimum IC50 concentration, the most effective antibodies were tested in double and triple combinations. Among the doubles, the combinations of N6+PGDM1400 and N6+PGT128 with IC50 ≤ 0.3 µg/mL proved to be the most effective. The most effective triple combination was N6+PGDM1400+PGT128. Our data demonstrate that this combination neutralizes pseudoviruses of the global HIV-1 panel with IC50 ≤ 0.11 µg/mL and IC80 ≤ 0.25 µg/mL.

Regulation of rat dopamine beta-hydroxylase gene transcription by early growth response gene 1 (Egr1).

Egr1, a transcription factor rapidly induced by various stimuli including stress, can elevate transcription of genes for the catecholamine biosynthetic enzymes TH and PNMT. To examine if Egr1 also regulates dopamine beta-hydroxylase (DBH) gene expression, PC12 cells were transfected with expression vector for full length or truncated inactive Egr1 and various DBH promoter-driven luciferase constructs. While Egr1 elevated TH promoter activity, DBH promoter activity was reduced. The reduction occurred as early as 4 h and reached maximal inhibition 16-40 h after transfection. Egr1 also reduced the expression of endogenous DBH mRNA and the induction of DBH promoter activity by cAMP. These effects were not observed with truncated Egr1 lacking the DNA binding domain. The first 247, but not 200, nucleotides of DBH promoter are sufficient for this suppression. Several putative Egr1 motifs were identified, and mutagenesis showed that the motif at -227/-224 is required. Binding of Egr1 to this region of the DBH promoter was verified by chromatin immunoprecipitation and electrophoretic mobility shift assays. This study demonstrates that DBH promoter contains at least one functional Egr1 motif; and indicates, for the first time, that Egr1 can play an inhibitory role in regulation of DBH gene transcription.

Optimization of Polycistronic Anti-CCR5 Artificial microRNA Leads to Improved Accuracy of Its Lentiviral Vector Transfer and More Potent Inhibition of HIV-1 in CD4⁺ T-Cells.

C-C chemokine receptor type 5 (CCR5) is utilized by human immunodeficiency virus (HIV) as a co-receptor for cell entry. Suppression of the CCR5 gene by artificial microRNAs (amiRNAs) could confer cell resistance. In previous work, we created a lentivector that encoded the polycistron of two identical amiRNAs that could effectively suppress CCR5. However, tandem repeats in lentiviral vectors led to deletions of the repeated sequences during reverse transcription of the vector RNA. To solve this problem, we have created a new amiRNA against CCR5, mic1002, which has a different microRNA scaffold and targets a different sequence. Replacing one of the two identical tandem amiRNAs in the polycistron with the mic1002 amiRNA increased the accuracy of its lentiviral vector transfer while retaining its ability to effectively suppress CCR5. A lentiviral vector containing two heterogenic amiRNAs significantly inhibited HIV replication in a vector-transduced human CD4⁺ lymphocyte culture.

Effect of prolonged nicotine infusion on response of rat catecholamine biosynthetic enzymes to restraint and cold stress.

There is a paradoxical relationship between nicotine and stress. To help elucidate their relationship on catecholamine biosynthesis, rats were infused with nicotine for 7-14 days before exposure to cold or restraint stress. Nicotine (5 mg/kg/day, 14 days) did not alter basal plasma corticosterone or its elevation with 24 h cold stress, but prevented corticosterone elevation following 2 h restraint stress. In adrenal medulla (AM), response of dopamine beta-hydroxylase (DBH), but not tyrosine hydroxylase (TH) mRNA, to both stressors was attenuated in nicotine-infused rats. In locus coeruleus (LC), restraint stress elevated TH and DBH mRNA in saline-, but not in nicotine-infused rats. Cold stress triggered a similar response of TH and DBH mRNAs in LC with and without nicotine infusion. With shorter nicotine infusion (8 mg/kg/day, 7 days), TH mRNA in AM was not induced by restraint stress on one (1x) or two (2x) consecutive days nor was DBH mRNA in AM or LC by 2x. The findings demonstrate that constant release of nicotine can modulate, or even prevent, some stress responses at the level of the HPA axis and gene expression of catecholamine biosynthetic enzymes in LC and AM.