rAAV expressing recombinant antibody for emergency prevention and long-term prophylaxis of COVID-19

IntroductionNumerous agents for prophylaxis of SARS-CoV-2-induced diseases are currently registered for the clinical use. Formation of the immunity happens within several weeks following vaccine administration which is their key disadvantage. In contrast, drugs based on monoclonal antibodies, enable rapid passive immunization and therefore can be used for emergency pre- and post-exposure prophylaxis of COVID-19. However rapid elimination of antibody-based drugs from the circulation limits their usage for prolonged pre-exposure prophylaxis.MethodsIn current work we developed a recombinant adeno-associated viral vector (rAAV), expressing a SARS-CoV-2 spike receptor-binding domain (RBD)-specific antibody P2C5 fused with a human IgG1 Fc fragment (P2C5-Fc) using methods of molecular biotechnology and bioprocessing.Results and discussionsA P2C5-Fc antibody expressed by a proposed rAAV (rAAV-P2C5-Fc) was shown to circulate within more than 300 days in blood of transduced mice and protect animals from lethal SARS-CoV-2 virus (B.1.1.1 and Omicron BA.5 variants) lethal dose of 105 TCID50. In addition, rAAV-P2C5-Fc demonstrated 100% protective activity as emergency prevention and long-term prophylaxis, respectively. It was also demonstrated that high titers of neutralizing antibodies to the SARS-CoV-2 virus were detected in the blood serum of animals that received rAAV-P2C5-Fc for more than 10 months from the moment of administration.Our data therefore indicate applicability of an rAAV for passive immunization and induction of a rapid long-term protection against various SARS-CoV-2 variants

Safety and immunogenicity of rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine against SARS-CoV-2 in healthy adolescents: an open-label, non-randomized, multicenter, phase 1/2, dose-escalation study

To protect young individuals against SARS-CoV-2 infection, we conducted an open-label, prospective, non-randomised dose-escalation Phase 1/2 clinical trial to evaluate the immunogenicity and safety of the prime-boost “Sputnik V” vaccine administered at 1/10 and 1/5 doses to adolescents aged 12–17 years. The study began with the vaccination of the older cohort (15-to-17-year-old participants) with the lower (1/10) dose of vaccine and then expanded to the whole group (12-to-17-year-old participants). Next, 1/5 dose was used according to the same scheme. Both doses were well tolerated by all age groups. No serious or severe adverse events were detected. Most of the solicited adverse reactions were mild. No significant differences in total frequencies of adverse events were registered between low and high doses in age-pooled groups (69.6% versus 66.7%). In contrast, the 1/5 dose induced significantly higher humoral and T cell-mediated immune responses than the 1/10 dose. The 1/5 vaccine dose elicited higher antigen-binding (both S and RBD-specific) as well as virus-neutralising antibody titres at the maximum of response (day 42), also resulting in a statistically significant difference at a distanced timepoint (day 180) compared to the 1/10 vaccine dose. Higher dose resulted in increased cross-neutralization of Delta and Omicron variants.;Clinical Trial RegistrationClinicalTrials.gov, NCT04954092, LP-007632

Genetic Passive Immunization with Adenoviral Vector Expressing Chimeric Nanobody-Fc Molecules as Therapy for Genital Infection Caused by Mycoplasma hominis.

Developing pathogen-specific recombinant antibody fragments (especially nanobodies) is a very promising strategy for the treatment of infectious disease. Nanobodies have great potential for gene therapy application due to their single-gene nature. Historically, Mycoplasma hominis has not been considered pathogenic bacteria due to the lack of acute infection and partially due to multiple studies demonstrating high frequency of isolation of M. hominis samples from asymptomatic patients. However, recent studies on the role of latent M. hominis infection in oncologic transformation, especially prostate cancer, and reports that M. hominis infects Trichomonas and confers antibiotic resistance to Trichomonas, have generated new interest in this field. In the present study we have generated specific nanobody against M. hominis (aMh), for which the identified target is the ABC-transporter substrate-binding protein. aMh exhibits specific antibacterial action against M. hominis. In an attempt to improve the therapeutic properties, we have developed the adenoviral vector-based gene therapy approach for passive immunization with nanobodies against M. hominis. For better penetration into the mucous layer of the genital tract, we fused aMh with the Fc-fragment of IgG. Application of this comprehensive approach with a single systemic administration of recombinant adenovirus expressing aMh-Fc demonstrated both prophylactic and therapeutic effects in a mouse model of genital M. hominis infection

Hypothetical mechanism of blocking nutrient acquisition through ABC transporter with aMh-FcG2a.

<p>Hypothetical mechanism of blocking nutrient acquisition through ABC transporter with aMh-FcG2a.</p

rAd5-CMV-PLAP-aMh-FcG2a and control rAd5-CMV-PLAP-aMh-ILZ-HA constructions and purification.

<p>(A) Scheme of expression cassettes for rAd5-CMV constructions; (B) sizing of purified Ad5-CMV-PLAP-aMh-FcG2a corresponding to virions suspensions with a particle size of ~100 nM.</p

Titers of <i>M</i>. <i>hominis</i> in vaginal washes with “prophylactic” scheme of rAds inoculation.

<p>Vaginal washes were collected 5 days after <i>M</i>. <i>hominis</i> inoculation (6 days after rAds inoculation). Amount of <i>M</i>. <i>hominis</i> was evaluated with real-time PCR. The rAd5-CMV-PLAP-aMh-FcG2a group exhibited a significantly lower <i>M</i>. <i>hominis</i> titer (Student's t-test = 3.5; p<0.01). Ad-null n = 5, PBS n = 15, rAd5-CMV-PLAP-aMh-FcG2a n = 10, rAd5-CMV-PLAP-aMh-ILZ-HA n = 11. In the “prophylactic” scheme of rAds inoculation, the <i>M</i>. <i>hominis</i> titer was significantly decreased in the rAd5-CMV-PLAP-aMh-FcG2a group. The prophylactic scheme, however, is not practical for treating mycoplasma infection. In the “therapeutic” scheme, the number of animals diagnosed as positive at 7 days after inoculation with rAd5-CMV-PLAP-aMh-FcG2a (12 days after <i>M</i>. <i>hominis</i> inoculation) was significantly lower than that of the other groups. Nevertheless the mycoplasma titer was not significantly different among the infected animals in any of the groups and not informative due to the large variability in counts and the low number of infected animals in the rAd5-CMV-PLAP-aMh-FcG2a group (n = 2 at 3 days and n = 1 at 7 days).</p

Determination of affinity constants.

<p>Binding of antigen to aMh-FcG2a was determined by surface plasmon resonance using Biacore 3000 (GE Healthcare). Antigen concentration series 5.95 nM; 11.9 nM; 59.5 nM and 595 nM (colour) and 1:1 fitting (black) interaction antigen to aMh-Fc. The fitted constant are k_a = 1.78⁴ M^-1s^-1 and k_d = 1.06⁻⁴ s^-1 which results <i>K</i>_<i>D</i> = 5.94*10⁻⁹ M (R_max = 428 RU; chi² = 11.8). Evaluation included double reference subtraction.</p

Dimer formations of aMh-FcG2a.

<p>Protein samples aMh-FcG2a were prepared before electrophoresis in sample buffer with or without DTT (as reducing agent), canonical mouse IgG2a was prepared with DTT and used as control. Protein bands ~42 and ~84 kDa corresponding monomer and dimer forms of aMh-Fc; protein bands ~54 kDa and 25 kDa corresponding heavy and light chains of canonical mouse IgG2a.</p

Revaccination in Age-Risk Groups with Sputnik V Is Immunologically Effective and Depends on the Initial Neutralizing SARS-CoV-2 IgG Antibodies Level

Vaccination against COVID-19 has occurred in Russia for more than two years. According to the Russian official clinical guidelines to maintain tense immunity in the conditions of the ongoing COVID-19 pandemic, it is necessary to use booster immunization six months after primary vaccination or a previous COVID-19 contraction. It is especially important to ensure the maintenance of protective immunity in the elderly, who are at risk of severe courses of COVID-19. Meanwhile, the immunological effectiveness of the booster doses has not been sufficiently substantiated. To investigate the immunogenicity of Sputnik V within the recommended revaccination regimen and evaluate the effectiveness of booster doses, we conducted this study on 3983 samples obtained from individuals previously vaccinated with Sputnik V in Moscow. We analyzed the level of antibodies in BAU/mL three times: (i) six months after primary immunization immediately before the booster (RV), (ii) 3 weeks after the introduction of the first component of the booster (RV1), and (iii) 3 weeks after the introduction of the second component of the booster (RV2). Six months after the primary vaccination with Sputnik V, 95.5% of patients maintained a positive level of IgG antibodies to the receptor-binding domain (RBD) of SARS-CoV-2. The degree of increase in the specific virus-neutralizing antibodies level after revaccination increased with a decrease in their initial level just before the booster dose application. In the group of people with the level of antibodies up to 100 BAU/mL six months after the vaccination, a more than eightfold increase (p p p p < 0.05), regardless of the previous COVID-19 infection. Thus, revaccination is most effective in individuals with an antibody level below 500 BAU/mL, regardless of the vaccinee age and COVID-19 contraction. For the first time, it has been shown that a single booster dose of the Sputnik vaccine is sufficient to form a protective immunity in most vaccinees regardless of age and preexisting antibody level

Vaccine formulations containing a combination of TLR4 and NOD2 agonists significantly enhance maturation of BMDC compared to formulations containing individual PRR agonists.

<p>BMDCs were harvested on day 8 of culture and incubated in 24-well plates for 24 hours with vaccine formulations. Expression of the maturation markers, CD80 <b>(A)</b>, CD86 <b>(B)</b>, and major histocompatibility complex (MHC) class II <b>(C)</b> was assessed by flow cytometric analysis of 5x10⁴ CD11c+ cells. Mean MFI values (indicating expression level) ± SEM from two independent experiments with 5 replicates each are shown. * indicates significant difference (P≤0.05) between formulations containing MDP or MPLA individually and the formulation without PRR agonists. # indicates significant difference (P≤0.05) between Alum+OVA+MDP+MPLA treatment and Alum+OVA+MPLA or Alum+OVA+MDP (Student’s t-test). <b>(D)</b> Cytokine levels were measured in cell-free culture supernatants collected 24 hours after addition of vaccine formulations to BMDCs (4x10⁴ cells/well) using bead-based immunoassay. Data represent mean ± SD. * indicates significant difference (P≤0.05) between formulations containing MDP or MPLA individually and the formulation without PRR agonists. # indicates significant difference (P≤0.05) between Alum+OVA+MDP+MPLA treatment and Alum+OVA+MPLA or Alum+OVA+MDP (Student’s t-test).</p