An Alphavirus -derived replicon RNA vaccine induces SARS-CoV-2 neutralizing antibody and T cell responses in mice and nonhuman primates

In the paper

“The S protein mediates binding of coronaviruses to angiotensin converting enzyme 2 (ACE2) on the surface of various cell types including epithelial cells of the pulmonary alveolus (4–6).”

What the sources say

• ?Not checked — no full text of the sourcecites [4] Hamming I., Timens W., Bulthuis M. L. C., Lely A. T., Navis G., van Goor H., Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J. Pathol. 203, 631– 637 (2004). 10.1002/path.1570 15141377 PMC7167720 source
• Partly supported

  The Results (‘Receptor usage of SARS-CoV-2’; page not provided) report that a spike chimera containing the SARS-CoV-2 receptor-binding domain entered cells expressing human ACE2. The paper does not establish ACE2 binding on pulmonary alveolar epithelial cells. Its Main section also identifies a different receptor for MERS-CoV, so the ACE2 statement does not apply to coronaviruses generally. (LLM, conf=0.87)

  cites [5] Letko M., Marzi A., Munster V., Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat. Microbiol. 5, 562– 569 (2020). 10.1038/s41564-020-0688-y 32094589 PMC7095430 sourceExperimental — accuracy not yet measured
• Partly supported

  The Introduction and Results section “ACE2 Is an Entry Receptor for SARS-CoV-2” establish that SARS-CoV-2 spike binds ACE2 and can mediate entry into ACE2-expressing cells. The same Results section identifies ACE2 at the surface of type II pneumocytes—alveolar epithelial cells—but makes that statement about SARS-CoV, not SARS-CoV-2. The cited paper therefore does not directly establish SARS-CoV-2 spike binding on those lung cells. (LLM, conf=0.84)

  cites [6] Walls A. C., Park Y.-J., Tortorici M. A., Wall A., McGuire A. T., Veesler D., Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 181, 281– 292.e6 (2020). 10.1016/j.cell.2020.02.058 32155444 PMC7102599 sourceExperimental — accuracy not yet measured

In the paper

“As a result, repRNA acts as its own adjuvant, eliciting more robust immune responses after a single dose, relative to conventional mRNA which typically requires multiple, 1,000-fold higher doses (29).”

What the sources say

• Partly supported

  The Discussion (page unavailable) says self-amplifying RNA provides additional immune stimuli, consistent with the adjuvant-like part of the claim. But the Results report equivalent protection at a 64-fold lower dose, not a 1,000-fold lower dose, and Figure 1 describes a prime-boost regimen for both RNA vaccines, not a single dose of self-amplifying RNA versus multiple doses of conventional mRNA. (LLM, conf=0.91)

  cites [29] Vogel A. B., Lambert L., Kinnear E., Busse D., Erbar S., Reuter K. C., Wicke L., Perkovic M., Beissert T., Haas H., Reece S. T., Sahin U., Tregoning J. S., Self-Amplifying RNA Vaccines Give Equivalent Protection against Influenza to mRNA Vaccines but at Much Lower Doses. Mol. Ther. 26, 446– 455 (2018). 10.1016/j.ymthe.2017.11.017 29275847 PMC5835025 sourceExperimental — accuracy not yet measured

In the paper

“A recent study indicates SARS-CoV-2-specific T cell responses may be associated with better recovery from COVID-19 (57).”

What the sources say

• Partly supported

  The Results (CD4+ and CD8+ T Cell Responses; page unavailable) report SARS-CoV-2-specific T cell responses in recovered COVID-19 patients. The study did not compare patients with better and worse recovery, so it does not establish an association between those responses and better recovery. (LLM, conf=0.85)

  cites [57] Grifoni A., Weiskopf D., Ramirez S. I., Mateus J., Dan J. M., Moderbacher C. R., Rawlings S. A., Sutherland A., Premkumar L., Jadi R. S., Marrama D., de Silva A. M., Frazier A., Carlin A. F., Greenbaum J. A., Peters B., Krammer F., Smith D. M., Crotty S., Sette A., Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals. Cell 181, 1489– 1501.e15 (2020). 10.1016/j.cell.2020.05.015 32473127 PMC7237901 sourceExperimental — accuracy not yet measured

In the paper

“Our findings demonstrate that LION/repRNA-CoV2S elicited modest but detectable T cell responses in macaques (Fig. 5), an outcome that is consistent with previous studies of SARS-CoV-2 S-targeted DNA and live viral vector vaccines tested in nonhuman primates (53, 58).”

What the sources say

• Supported

  In “Construction and immunogenicity of DNA vaccine candidates,” the paper reports detectable S-specific cellular and CD4+ and CD8+ T-cell responses in vaccinated rhesus macaques. This supports the comparison to a previous S-targeted DNA vaccine study; the citing authors’ LION/repRNA-CoV2S result is their own finding, not one attributed to this source. (LLM, conf=0.93)

  cites [53] Yu J., Tostanoski L. H., Peter L., Mercado N. B., McMahan K., Mahrokhian S. H., Nkolola J. P., Liu J., Li Z., Chandrashekar A., Martinez D. R., Loos C., Atyeo C., Fischinger S., Burke J. S., Slein M. D., Chen Y., Zuiani A., N Lelis F. J., Travers M., Habibi S., Pessaint L., Van Ry A., Blade K., Brown R., Cook A., Finneyfrock B., Dodson A., Teow E., Velasco J., Zahn R., Wegmann F., Bondzie E. A., Dagotto G., Gebre M. S., He X., Jacob-Dolan C., Kirilova M., Kordana N., Lin Z., Maxfield L. F., Nampanya F., Nityanandam R., Ventura J. D., Wan H., Cai Y., Chen B., Schmidt A. G., Wesemann D. R., Bari source
• Supported

  The Immunogenicity in rhesus macaques section reports spike-specific T-cell responses after vaccination with ChAdOx1 nCoV-19, a viral-vector vaccine. This supports the cited comparison with prior nonhuman-primate vaccine findings; the sentence attributes the LION/repRNA-CoV2S result to the citing authors’ own experiment. (LLM, conf=0.90)

  cites [58] N. Van Doremalen, T. Lambe, A. Spencer, S. Belij-rammerstorfer, J. N. Purushotham, J. R. Port, V. Avanzato, T. Bushmaker, M. Ulaszewska, F. Feldmann, E. R. Allen, H. Sharpe, J. Schulz, M. Holbrook, A. Okumura, K. Meade-white, C. Bissett, C. Gilbride, B. N. Williamson, R. Rosenke, D. Long, A. Ishwarbhai, R. Kailath, L. Rose, S. Morris, C. Powers, J. Lovaglio, P. W. Hanley, D. Scott, G. Saturday, E. De Wit, C. Sarah, V. J. Munster, ChAdOx1 nCoV-19 vaccination prevents SARS-CoV-2 pneumonia in rhesus macaques. bioRxiv 10.1101/2020.05.13.093195 (2020). source

In the paper

“In the former report, IC50 titers as low as 1:8 were associated with protection from re-infection while in the latter, IC50 titers as low as 1:50 were associated with reduced viral load and protection from lung pathology.”

What the sources say

• Partly supported

  The ‘former report’ is this paper; the ‘latter’ finding is attributed to a different report and is not assessed here. In ‘SARS-CoV-2-infection protect monkeys from reinfection’ (page unavailable), the paper reports no detectable viral load after rechallenge and suggests neutralizing antibodies might protect against reinfection. Our copy does not establish the specific IC50 titer of 1:8; the Table 1 text is garbled. (LLM, conf=0.82)

  cites [51] L. Bao, W. Deng, H. Gao, C. Xiao, J. Liu, J. Xue, Q. Lv, J. Liu, P. Yu, Y. Xu, F. Qi, Y. Qu, F. Li, Z. Xiang, H. Yu, S. Gong, M. Liu, G. Wang, S. Wang, Z. Song, W. Zhao, Y. Han, L. Zhao, X. Liu, Q. Wei, C. Qin, Reinfection could not occur in SARS-CoV-2 infected rhesus macaques, bioRxiv, 2020.03.13.990226 (2020). sourceExperimental — accuracy not yet measured
• Supported

  The citation to this paper concerns the latter report’s 1:50 finding, not the former report’s 1:8 finding. In the macaque challenge study (section labeled Abstract; page unavailable), pre-challenge neutralizing-antibody titers were about 50, and vaccinated animals had significantly lower viral loads and only mild, focal lung changes, compared with severe pneumonia in controls. (LLM, conf=0.90)

  cites [52] Gao Q., Bao L., Mao H., Wang L., Xu K., Yang M., Li Y., Zhu L., Wang N., Lv Z., Gao H., Ge X., Kan B., Hu Y., Liu J., Cai F., Jiang D., Yin Y., Qin C., Li J., Gong X., Lou X., Shi W., Wu D., Zhang H., Zhu L., Deng W., Li Y., Lu J., Li C., Wang X., Yin W., Zhang Y., Qin C., Development of an inactivated vaccine candidate for SARS-CoV-2. Science 369, 77– 81 (2020). 10.1126/science.abc1932 32376603 PMC7202686 source

In the paper

“Following natural infection of humans with the related SARS-CoV, neutralizing antibody and memory B cell responses in some individuals are reported to be short lived (~ 3 years), whereas memory T cells persist for at least 6 years (54–56)”

What the sources say

• ?Not checked — no full text of the sourcecites [54] Tang F., Quan Y., Xin Z.-T., Wrammert J., Ma M.-J., Lv H., Wang T.-B., Yang H., Richardus J. H., Liu W., Cao W.-C., Lack of peripheral memory B cell responses in recovered patients with severe acute respiratory syndrome: A six-year follow-up study. J. Immunol. 186, 7264– 7268 (2011). 10.4049/jimmunol.0903490 21576510 source
• Partly supported

  The Introduction reports that SARS-specific memory T cells persisted for up to six years after recovery, and the Conclusion reports their detection at nine and eleven years. The Introduction also says SARS-specific antibody levels were undetectable at six years, but our copy does not establish that neutralizing antibodies or memory B cells were short-lived at approximately three years. (LLM, conf=0.86)

  cites [55] Ng O.-W., Chia A., Tan A. T., Jadi R. S., Leong H. N., Bertoletti A., Tan Y.-J., Memory T cell responses targeting the SARS coronavirus persist up to 11 years post-infection. Vaccine 34, 2008– 2014 (2016). 10.1016/j.vaccine.2016.02.063 26954467 PMC7115611 sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [56] Chen H., Hou J., Jiang X., Ma S., Meng M., Wang B., Zhang M., Zhang M., Tang X., Zhang F., Wan T., Li N., Yu Y., Hu H., Yang R., He W., Wang X., Cao X., Response of memory CD8+ T cells to severe acute respiratory syndrome (SARS) coronavirus in recovered SARS patients and healthy individuals. J. Immunol. 175, 591– 598 (2005). 10.4049/jimmunol.175.1.591 15972696 source

In the paper

“anti-S T-cell responses to the related SARS- and MERS-CoVs contribute toward viral clearance in normal as well as aged mice infected with SARS- or MERS-CoV, respectively (43–45).”

What the sources say

• Partly supported

  The Results identify S366 as an epitope of the SARS-CoV spike protein and report that transferring S366-specific CD8 T cells accelerated viral clearance in infected BALB/c mice. This supports the SARS-CoV portion of the claim, but the paper does not establish the claimed effect in aged mice or in MERS-CoV-infected mice. (LLM, conf=0.88)

  cites [43] Zhao J., Zhao J., Perlman S., T cell responses are required for protection from clinical disease and for virus clearance in severe acute respiratory syndrome coronavirus-infected mice. J. Virol. 84, 9318– 9325 (2010). 10.1128/JVI.01049-10 20610717 PMC2937604 sourceExperimental — accuracy not yet measured
• Partly supported

  The sections on T-cell responses in infected animals and on memory T cells report that spike-specific responses contribute to SARS-CoV clearance in young mice and can improve clearance or protection in older mice. The MERS-CoV passage reports spike-specific CD8 T cells and a role for T cells in clearance in susceptible mice, but does not establish the claimed effect in aged MERS-CoV-infected mice. Page numbers are unavailable in our copy. (LLM, conf=0.86)

  cites [44] Channappanavar R., Zhao J., Perlman S., T cell-mediated immune response to respiratory coronaviruses. Immunol. Res. 59, 118– 128 (2014). 10.1007/s12026-014-8534-z 24845462 PMC4125530 sourceExperimental — accuracy not yet measured
• Partly supported

  The Results report that responses to the SARS-CoV spike epitopes S436 and S525 accelerated viral clearance in 8- to 10-month-old mice. The Discussion says virus-specific CD8 T cells are important for MERS-CoV clearance, but does not establish that those cells target spike or that the finding applies to both young and aged MERS-CoV-infected mice. (LLM, conf=0.87)

  cites [45] Channappanavar R., Fett C., Zhao J., Meyerholz D. K., Perlman S., Virus-specific memory CD8 T cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection. J. Virol. 88, 11034– 11044 (2014). 10.1128/JVI.01505-14 25056892 PMC4178831 sourceExperimental — accuracy not yet measured

In the paper

“A prime-only replicating RNA vaccine delivered with lipid inorganic nanoparticles induces neutralizing antibody and T cell responses against SARS-CoV-2 in aged mice and macaques.”

In the paper

“Mice receiving a 10 or 1 μg prime/boost exhibited robust splenic T cell responses with mean IFN-γ spots/106 cells of 1179, 1328, and 946 in the 10 μg groups and 458, 409, and 1231 in the 1 μg groups for the 17-, 8-, and 2-month old mice, respectively (Fig. 3B).”

In the paper

“LION maintained full integrity of the repRNA molecules (Fig. 1H) and complex size (Fig. 1I) at all time points.”

In the paper

“However, following a boost immunization, the 17-month old mice developed significantly higher antibody responses relative to their prime responses (p=0.001 and p<0.0001, for the 10 and 1 μg doses, respectively), approaching the post-boost responses seen in the 8- and 2-month old mice; antibody responses in the 17-month old 1 μg dose group were still significantly lower than the 2-month-old”

In the paper

“All three dose groups, 10, 1, and 0.1 μg, responded to a boost immunization, reaching anti-S mean IgG concentrations of 1517, 937, and 312 μg/ml, respectively (Fig. 2A).”

In the paper

“No significant differences were observed between age groups, however, more variability in T cell responses were observed in the 17-month old mice relative to the 8- and 2-month old mice.”

In the paper

“Both the 10 and 1 μg prime-only doses induced neutralizing antibodies with mean 50% inhibitory concentrations (IC50) of 1:643 and 1:226, respectively, significantly higher than titers induced by the 0.1 μg dose (p=0.03 and p=0.02, respectively)”

In the paper

“When mixed, electrostatic association between anionic repRNA and cationic DOTAP molecules on the surface of LION promotes immediate complex formation, as confirmed by an increase in particle size to an intensity-weighted average diameter of 90 nm detected by dynamic light scattering (Fig. 1F).”

In the paper

“Robust T cell responses were also detected in the lung and were similar between groups with mean IFN-γ spots/106 cells of 756, 784, and 777, respectively (Fig. 2F).”

In the paper

“partial seroconversion (2 out of 5) at a 0.1 μg dose, increasing to 4 out of 5 by day 28 (Fig. 2A).”

In the paper

“analysis of the specificity of peptide response showed a biased response toward the S1 domain of the S protein in the spleen (Fig. S1A), whereas responses in the lung were more broadly distributed between the S1 and S2 domains of the S protein (Fig. S1B).”

In the paper

“LION/repRNA-CoV2S induced ratios of IgG2a:IgG1 greater than 1 (Fig. 3C, D) in all age groups of BALB/c mice, indicating a Th1-biased immune response.”

In the paper

“all three macaques immunized with the prime-only 250 μg dose seroconverted as early as day 10, with anti-S IgG concentrations continuing to increase in these 3 animals to 48, 51, and 61 μg/ml by day 42, then appearing to plateau through at least day 70 post vaccination (Fig. 4B).”

In the paper

“Here, we show that the LION/repRNA-CoV2S vaccine induced robust neutralization of SARS-CoV-2 in vaccinated mice and pigtail macaques, with a mean PRNT80 titer in all 5 macaques of 1:197.”

In the paper

“Peak responses varied between days 28-42 and were independent of the vaccine dose.”

In the paper

“None of the animals became severely ill during the course of the study and none required euthanasia.”

In the paper

“sera from the three macaques immunized with the prime-only 250 μg dose neutralized pseudovirus (SARS-CoV-2 Wuhan-Hu-1 pseudotype) transduction of cells in vitro with reciprocal IC50 titers of 1:38, 1:20 and 1:47 by day 28, with IC50 titers increasing to 1:472, 1:108, and 1:149 by day 42; the 50 μg dose group achieved similar robust IC50 titers only after the booster immunization, reaching pseudov”

In the paper

“By day 42, mean PRNT80 titers significantly increased to 1:176 after a booster immunization in the 50 μg dose group and to 1:211 in the prime-only 250 μg dose group, (Fig. 4D, p=0.012 for both doses, and Fig. S4).”

In the paper

“Frequencies of IFN-γ producing CD4+ or CD8+ T cells showed an increasing trend in some animals by 42 days post vaccination, although changes were not significant (Fig. 5C).”

In the paper

“all 5 macaques developed PRNT80 titers within the same range as titers measured in the seven convalescent humans (<1:20 to 1:1280, collected 15 to 64 days post onset of symptoms) and there was no significant difference in mean neutralizing titers between all 5 vaccinated macaques (1:197) and convalescent humans (1:518) (P=0.27, Fig. 4D, Fig. S4, and Table S1).”

In the paper

“Both macaques receiving 50 μg repRNA-CoV2S seroconverted after a single dose but developed lower antibody responses with anti-S IgG concentrations of 1 and 0.5 μg/ml by day 28, compared to 7, 20, and 45 μg/ml in the 250 μg dose group at this same time point (Fig. 4B).”

In the paper

“repRNA vaccines mimic an alphavirus infection in that viral-sensing stress factors are triggered and innate pathways are activated through Toll-like receptors and retinoic acid inducible gene (RIG)-I to produce interferons, pro-inflammatory factors and chemotaxis of antigen-presenting cells, as well as promoting antigen cross-priming (28).”

What the sources say

• ?Not checked — no full text of the sourcecites [28] Jensen S., Thomsen A. R., Sensing of RNA viruses: A review of innate immune receptors involved in recognizing RNA virus invasion. J. Virol. 86, 2900– 2910 (2012). 10.1128/JVI.05738-11 22258243 PMC3302314 source

In the paper

“introduction of repRNA into cells initiates ongoing biosynthesis of antigen-encoding RNA that results in dramatically increased expression and duration that enhances humoral and cellular immune responses (27).”

What the sources say

• ?Not checked — no full text of the sourcecites [27] Berglund P., Smerdou C., Fleeton M. N., Tubulekas I., Liljeström P., Enhancing immune responses using suicidal DNA vaccines. Nat. Biotechnol. 16, 562– 565 (1998). 10.1038/nbt0698-562 9624688 source

In the paper

“Endpoints were selected prior to the start of each study and were selected based on the primary objective of characterizing the safety and immune responses to vaccination with a LION/repRNA-CoV2S vaccine.”

In the paper

“IgG2c isotype was selected for C57BL/6 sera due to their lack of IgG2a isotype (64)”

What the sources say

• ?Not checked — no full text of the sourcecites [64] Martin R. M., Brady J. L., Lew A. M., The need for IgG2c specific antiserum when isotyping antibodies from C57BL/6 and NOD mice. J. Immunol. Methods 212, 187– 192 (1998). 10.1016/S0022-1759(98)00015-5 9672206 source

In the paper

“Group sizes in the macaque study were limited by availability of animals.”

In the paper

“while IgG2a is utilized for BALB/C sera (65).”

What the sources say

• ?Not checked — no full text of the sourcecites [65] Aberle J. H., Aberle S. W., Kofler R. M., Mandl C. W., Humoral and cellular immune response to RNA immunization with flavivirus replicons derived from tick-borne encephalitis virus. J. Virol. 79, 15107– 15113 (2005). 10.1128/JVI.79.24.15107-15113.2005 16306582 PMC1316042 source

In the paper

“No blinding was used throughout all studies.”

In the paper

“This is most likely due to the sustained antigen expression mediated by repRNA, which may result in plasma cell activation akin to what happens following traditional vaccine booster immunizations.”

In the paper

“However, because the 250 μg dose was administered concurrently to five injection sites in the pigtail macaques, it cannot be ruled out that immunogenicity and safety measures would be different to those following administration of this dose via a single bolus injection.”

In the paper

“However, these conclusions are drawn in the absence of challenge data and from a limited number of animals and at time points after vaccination where observed responses, especially T-cell responses, exhibited great variability.”

In the paper

“Mouse group sizes were based on power analyses using data from previous experiments using a similar repRNA platform and mice were randomly distributed between groups.”

In the paper

“Five pigtail macaques were stratified based on age and weight.”