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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   May 29, 2022

   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.	A2 suggested: None
   The 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 ( M suggested: None
   The 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: None nucleocapsid antigen ( N ) with anti-nucleocapsid antibodies ( B2) suggested: None
   To 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-complex suggested: None anti-human IgM suggested: None
   Six weeks later, all vaccinated animals were bled and, from their sera, anti-complex precipitated globulin antibodies were collected.	anti-complex precipitated globulin suggested: None
   Each group of rabbits produced 15 ml of immune serum (200 mg) containing antibodies to the immune complexes A1, A2, A3, B1, or B2.	B2 suggested: None
   The 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-complexes suggested: None
   Component 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).	A1 suggested: None
   Also, from day zero, blood samples were taken on days 14, 28, 42 & 56 to measure serum immunological markers and anti-complexes antibodies.	anti-complexes suggested: None
   Sera optical density at 450 nm was measured and the levels of anti-CRCx3 and anti-CRCx2 neutralizing antibodies (NAbs) were evaluated.	anti-CRCx3 suggested: None anti-CRCx2 neutralizing antibodies ( NAbs ) suggested: None
   Increasing serum anti-ICs antibody titers in vaccinated mice was monitored from the beginning and after days 14, 28, 42 & 56 (ELISA).	anti-ICs suggested: (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-B1 suggested: None anti-spike suggested: None
   Then, 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).	IC2 suggested: None IC3 suggested: None
   CRCx 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: None
   The 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 similarity suggested: None
   The 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) [66 suggested: None
   The 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-IC suggested: None antigen/specific-antibody-IC suggested: None anti-SARS-Cov-2 suggested: None CRCx2 suggested: None
   However, 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-nucleocapsid suggested: None
   Conclusively, 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-spike suggested: None
   In 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 (CRCx2 suggested: None
   Experimental 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 E6 suggested: None
   The genomic content of Vero cells of each well with the minimal number of plaques was extracted for further molecular characterization.	Vero suggested: 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/c suggested: None
   Software and Algorithms
   Sentences	Resources
   (Lonza Bioscience) +L	Lonza Bioscience suggested: (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.	SPSS suggested: (SPSS, RRID:SCR_002865)

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   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).

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   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.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   Results from rtransparent:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
   • No protocol registration statement was detected.

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   Results from scite Reference Check: We found no unreliable references.

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   Version published to 10.1101/2022.05.27.493693v1 on bioRxiv

   May 27, 2022