Development of a Novel SARS-CoV-2 Immune Complex Vaccine Candidate (CRCx) with Broad Immune Responses: A Preclinical Trial in Animal Model
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Abstract
Background
The ongoing pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a serious threat to global public health and imposes a severe burden on the entire human population. Faced with a virus that can mutate its structure while immunity is incapacitated, a need to develop a universal vaccine that can boost immunity to coronaviruses is highly needed.
Design
Five formulations of two types (CRCx2 and CRCx3) of immune complexes with an immunogen adjuvant were evaluated in a mouse model as candidate SARS CoV-2 vaccines in a pretrial prior to clinical trials in humans. CRCx3 comprises 3 different formulas and CRCx2 comprises 2. Balb/c mice were vaccinated intraperitoneally on days 0/7 with a high or low dose of CRCx2 or on days 0/7/14 with a high, medium, or low dose of CRCx3 series, and their blood was sampled for serum antibody measurements. Mice were challenged with live virus after immunization with either vaccine to evaluate prophylaxis ability or treated with them after challenge to evaluate therapeutic ability on day 15. Immunological markers and histopathological studies as well as titration of neutralizing antibodies to the vaccines were evaluated and analyzed.
Results
CRCx 3 and CRCx 2 vaccine candidates induced elevated levels of positive neutralizing antibodies as well as a cellular immune response with safety, efficient productivity, and good genetic stability for vaccine manufacturing to provide protection against SARS-CoV-2 with relatively higher levels with the high dose CRCx2 candidate combination.
Conclusions
Highly efficient protection and therapeutic effect against SARS-CoV-2 were obtained with a double-dose immunization schedule spaced at 7-day intervals using injections 0.25 of or 0.40 ml of CRCx2 vaccine formulations with a 25-mm needle. These results support further evaluation of CRCx in a clinical trial on humans.
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SciScore for 10.1101/2022.05.27.493693: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: This study was performed in accordance with the guidelines for the care and use of laboratory animals published and approved by the Committee for Ethics on Animal Experiments and the Committee for Animal Biosafety Level 3 Research of the Egyptian Military Scientific Commission.
Euthanasia Agents: Doses were mixed with Fc of IgG mouse anti-Human IgM added as an adjuvant immunogen dissolved in human albumin, phosphate buffer, and sodium chloride without any stabilizers or preservatives.Sex as a biological variable For the production of different IC antibodies, twenty-four male rabbits were divided into six groups (numbered 1 through 6), each comprising four rabbits. Randomization Mice were … SciScore for 10.1101/2022.05.27.493693: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: This study was performed in accordance with the guidelines for the care and use of laboratory animals published and approved by the Committee for Ethics on Animal Experiments and the Committee for Animal Biosafety Level 3 Research of the Egyptian Military Scientific Commission.
Euthanasia Agents: Doses were mixed with Fc of IgG mouse anti-Human IgM added as an adjuvant immunogen dissolved in human albumin, phosphate buffer, and sodium chloride without any stabilizers or preservatives.Sex as a biological variable For the production of different IC antibodies, twenty-four male rabbits were divided into six groups (numbered 1 through 6), each comprising four rabbits. Randomization Mice were randomly divided into twelve groups, five mice per group, and classified into two categories. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources nucleocapsid antibody, and membrane antibody (by AMSBIO) were procured, diluted with 1 ml of PBS; i.e. (1/400, 1/200, 1/100, 1/50, 1/25) and incubated at room temperature for at least 2 hours. 1.6. Preparation of Immune Complexes: Two different modes of immune complexes formulation were used in which mixing of a specific concentration of an antigen with its relevant concentration of an antibody took place and named A1, A2, A3, B1, and B2, where A1, A2, and A3 constitute the CRCx3 series and B1 and B2 constitute the CRCx2 series. A2suggested: NoneThe first mode (a non-specific mixture composed of coronavirus antigens and their non-specific antibodies) included SARS-CoV-2 spike protein (S1 subunit) with anti-nucleocapsid antibodies (A1), nucleocapsid antigen (N) with anti-membrane antibodies (A2) and membrane antigen (M) with anti-spike antibodies (A3). S1 subunit ) with anti-nucleocapsid antibodies ( A1) , nucleocapsid antigen ( N ) with anti-membrane antibodies ( A2 ) and membrane antigen ( Msuggested: NoneThe second mode (a specific mix composed of coronavirus antigens and their specific antibodies) included spike antigen (S1) with anti-spike antibodies (B1), and nucleocapsid antigen (N) with anti-nucleocapsid antibodies (B2). spike antigen ( S1 ) with anti-spike antibodies ( B1)suggested: Nonenucleocapsid antigen ( N ) with anti-nucleocapsid antibodies ( B2)suggested: NoneTo finalize the complex preparations (A1, A2, A3, B1, and B2) for inducing anti-complex antibodies production in immunized rabbits, 3 ml of the adjuvant goat IgG anti-human IgM Fc (5 ng/ml) (by ABCAM, Cambridge, UK) was dissolved in human albumin and phosphate-buffered normal saline added to the remainder of the sediments and packaged in 1 ml vials of injectable solution with labels (See Supplement: Fig. 4). 1.7. anti-complexsuggested: Noneanti-human IgMsuggested: NoneSix weeks later, all vaccinated animals were bled and, from their sera, anti-complex precipitated globulin antibodies were collected. anti-complex precipitated globulinsuggested: NoneEach group of rabbits produced 15 ml of immune serum (200 mg) containing antibodies to the immune complexes A1, A2, A3, B1, or B2. B2suggested: NoneThe amounts of anti-complex antibodies precipitated from each rabbit’s sample are then estimated according to the different concentrations of injected complexes mixtures by ELISA. 1.9. ELISA Assay of Produced Immune Complexes Antibodies: For quantitative titration of anti-immune-complexes produced from experimental rabbits’ sera to the different immune complexes formulae, an ELISA assay was performed by coating each one of five 96-well plates with a specific immune complex of A1, A2, A3, B1, or B2. anti-immune-complexessuggested: NoneComponent B is an anti-complex-antibodies standard calibrator: 5, 10, 20, 30, 40, and 50 μg/ml (previously prepared from rabbits’ sera) for each of the immune complexes (A1, A2, A3, B1, or B2). A1suggested: NoneAlso, from day zero, blood samples were taken on days 14, 28, 42 & 56 to measure serum immunological markers and anti-complexes antibodies. anti-complexessuggested: NoneSera optical density at 450 nm was measured and the levels of anti-CRCx3 and anti-CRCx2 neutralizing antibodies (NAbs) were evaluated. anti-CRCx3suggested: Noneanti-CRCx2 neutralizing antibodies ( NAbs )suggested: NoneIncreasing serum anti-ICs antibody titers in vaccinated mice was monitored from the beginning and after days 14, 28, 42 & 56 (ELISA). anti-ICssuggested: (Kadrmas JL; J Cell Biol. 2004 Cat# ics, RRID:AB_2568207)The highest produced NAb was Anti-B1 produced against the immune complex of the spike protein and its specific anti-spike antibody. Anti-B1suggested: Noneanti-spikesuggested: NoneThen, the infected T-helper cell will send signals to excite the B-cells to produce a number of immune antibodies to attach to the viral antigens that are configured by the respiratory cells, thus forming a circulating immune complex that comprises coronavirus antigen (M, N, and S and its specific neutralizing antibody as IC1, IC2, and IC3). IC2suggested: NoneIC3suggested: NoneCRCx was formulated with twenty-five micrograms (25 μg) of different antigens including spike protein (S1 subunit), nucleocapsid (N), or membrane antigen (M) as well as forty micrograms (40 μg) of different antibodies including anti-nucleocapsid, anti-membrane or anti-spike (S1 subunit) antibodies. anti-nucleocapsid, anti-membrane or anti-spike (S1 subunit)suggested: NoneThe main aim of this novel immune peptide CRCx is to stimulate the CD8+ T-cells against the foreign antigenic non-complexes as well as any other hidden circulating immune complex form with any antigenic similarity (antigen/specific-antibodies) and to put the whole immunity system on alert and restoring its normal functions. antigenic similaritysuggested: NoneThe results also did not find the vaccine to cause antibody-dependent enhancement (ADE) [66] as all the data obtained in this study support the safety and immunogenicity of this candidate vaccine series. antibody-dependent enhancement (ADE) [66suggested: NoneThe antigen/nonspecific-antibody-IC vaccine series needs more experiments to validate our primary hypothesis that they may prompt more potent and more long-lasting protection against mutating versions of SARS-Cov-2 as results showed that within the time limit of this trial, the antigen/specific-antibody-IC vaccine series produced higher NAbs against the vaccine ICs. 1.4. Conclusion: The new immune complex (IC) anti-SARS-Cov-2 candidate vaccine CRCx is composed of 5 different combinations: a series of three antigen/nonspecific-antibody termed (CRCx3) and two antigen/specific-antibody termed (CRCx2). antigen/nonspecific-antibody-ICsuggested: Noneantigen/specific-antibody-ICsuggested: Noneanti-SARS-Cov-2suggested: NoneCRCx2suggested: NoneHowever, again, the highest titer in these groups was seen with the high dose CRCx3 subtype composed of spike and its nonspecific antibody of anti-nucleocapsid. anti-nucleocapsidsuggested: NoneConclusively, according to the results, the spike antigen/anti-spike specific-antibody combination of CRCx2 gives the highest immunogenicity against Covid-19 virus infection both as a prophylaxis and as a therapy. antigen/anti-spikesuggested: NoneIn addition, even though both vaccines were found to significantly reduce or abolish viral load and broncho-alveolar effects in animal models challenged with SARS-CoV-2 within 14 days after receiving the booster dose of the vaccines with no signs of pneumonia in histopathological sections of the virus-challenged animals after vaccination, a higher preference was found to the double-dose antigen-specific-antibody (CRCx2) series and/or those from both CRCx series with the spike protein antigen. antigen-specific-antibody (CRCx2suggested: NoneExperimental Models: Cell Lines Sentences Resources Vero E6 cells with an average population of 104 cells were cultured for 18–24 h in each well in the growth medium [Eagle’s Minimum Essential Medium (EMEM) Vero E6suggested: NoneThe genomic content of Vero cells of each well with the minimal number of plaques was extracted for further molecular characterization. Verosuggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)Experimental Models: Organisms/Strains Sentences Resources Trail 2: For 14 days, starting on the day 15 from primary immunization in trail 1, the BALB/c mice of groups A, B, C, D, E, and F were intramuscularly challenged in the upper leg with a daily dose/mouse of 0.25 ml (total of 108 IU) of live SARS-Cov-2 virus to which 5 IU of DNA polymerase was added. BALB/csuggested: NoneSoftware and Algorithms Sentences Resources (Lonza Bioscience) +L Lonza Biosciencesuggested: (Science Exchange, RRID:SCR_010620)Statistical Analysis: The SPSS software Version 25 was used to analyze the level of significance, using one-way ANOVA, paired t-test (2-tailed), and Pearson’s correlation methods. SPSSsuggested: (SPSS, RRID:SCR_002865)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
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- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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