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s protein vaccine



A number of preventive vaccines are being developed to counter the coronavirus illness 2019 pandemic. The main candidates have now been evaluated in nonhuman primates (NHPs) and human section 1 and/or section 2 scientific trials. A number of vaccines have already superior into section 3 efficacy trials, whereas others will accomplish that earlier than the tip of 2020. Right here, we summarize what is understood of the antibody and T cell immunogenicity of those vaccines in NHPs and people. To the extent doable, we evaluate how the vaccines have carried out, bearing in mind the usage of totally different assays to evaluate immunogenicity and inconsistencies in how the ensuing information are offered. We additionally assessment the end result of problem experiments with extreme acute respiratory syndrome coronavirus 2 in immunized macaques, whereas noting variations within the protocols used, together with however not restricted to the virus problem doses. Press releases on the outcomes of vaccine efficacy trials are additionally summarized.



The coronavirus illness 2019 (COVID-19) pandemic rages unabated and should proceed to take action till there’s a protected, efficient, and broadly used protecting vaccine. A number of vaccines to stop extreme acute respiratory syndrome coronavirus 2 (SARS-CoV-2) an infection and/or COVID-19 illness at the moment are progressing by preclinical testing and section 1/2a human trials, whereas some are already in section 2b/3 efficacy trials in and out of doors the US ( and period=vaccine&cntry=&state=&metropolis=&dist=) (Desk 1) (1–32). A number of of those mid- to late-stage vaccines are a part of the U.S. authorities’s Operation Warp Pace (OWS), which has been reviewed elsewhere (33–35). A number of vaccine candidates produced in China are additionally properly superior within the analysis and approval course of (1, 2, 12, 14, 15, 22–24, 29, 30). Part 1/2 trial information on the Russian-made Sputnik V vaccine have now been printed (28).

All of the vaccines are both primarily based solely on the viral Spike (S) protein, which is run by numerous strategies together with expression from nonreplicating adenoviruses and nucleic acid vectors or as recombinant proteins, or are inactivated viruses that embrace the S protein along with all different structural viral proteins (Desk 1). The vaccines are all primarily based on S proteins containing D614, which was the dominant pressure after they had been designed. A variant virus with G614 in its S protein has since emerged to dominance globally due to its higher transmissibility (36–38). Nevertheless, this D614G change doesn’t have an effect on sensitivity to neutralization by sera from contaminated or vaccinated individuals, or to neutralizing monoclonal antibodies (MAbs), and due to this fact will not be problematic for vaccine efficacy (36–38). A few of the more moderen papers reviewed under embrace neutralization information utilizing the G614 virus. All of the adenovirus, mRNA, and DNA vaccine candidates listed in Desk 1 contain full-length S proteins; variants with truncations of the transmembrane area and/or the cytoplasmic tail had been examined as comparators in two macaque research (4, 6). The recombinant proteins from Novavax and Clover Biopharmaceuticals are primarily based on full-length S proteins (9, 10, 12, 21). Many S protein constructs incorporate two proline substitutions within the S2 area (K986P and V987P) that stabilize the expressed trimer within the prefusion construction that’s thought of to be optimally immunogenic for the induction of neutralizing antibodies (NAbs), whereas minimizing non-NAb responses (39). The identical technique was used to stabilize the respiratory syncytial virus F (fusion) protein and enhance its immunogenicity (40, 41). In a single macaque examine of adenovirus serotype 26 (Ad26) virus variants, the NAb response to the two-proline mutant S protein was stronger than to different constructs that contained or lacked stabilizing modifications, truncations, or various chief sequences (4). A mouse immunogenicity experiment that additionally in contrast Ad26 virus variants led to an identical conclusion (42). Comparative experiments in mice led to the inclusion of the identical double proline change (and a furin cleavage website knockout) within the Sanofi Pasteur mRNA vaccine (13). Recombinant S protein immunogens with the identical mixture of mutations supplied the strongest safety in opposition to virus problem in a mouse mannequin, in comparison with proteins that lacked these stabilizing modifications (43). In a macaque comparability of DNA vaccines expressing numerous S protein constructs, the authors reported {that a} soluble S protein that contained the 2 proline substitutions along with a cleavage website knockout and a trimerization area (S.dTM.PP) was higher than the corresponding truncated S protein (S.dTM) at conferring safety from SARS-CoV-2 problem (6).

Experimentation and precedent due to this fact assist the usage of stabilizing modifications that keep the SARS-CoV-2 S protein trimer in its prefusion conformation. Nevertheless, the S protein expressed within the AstraZeneca/Oxford College ChAdOx1 nCoV-19 vaccine is a wild-type sequence that doesn’t embrace any stabilizing modifications (3, 16, 17). The Sputnik V rAd5 and rAd26 adenovirus vectors specific a full-length S protein, however the printed report doesn’t point out whether or not stabilizing mutations had been added (28). Equally, it was not acknowledged whether or not the S protein within the CanSinoBIO Ad5-nCoV vaccine was stabilized (23, 24). Whether or not the absence of stabilizing modifications impacts the efficiency of those numerous adenovirus vaccines will not be recognized.

The Pfizer/BioNTech BNT162b1 vaccine was primarily based on the S protein’s receptor-binding area (RBD), however its growth was terminated after section 1/2 trials, in favor of the BNT162b2 assemble that expresses the entire, stabilized S protein (Desk 1) (11, 25–27). All of the mRNA vaccines are encapsulated inside liposomes of unpublished composition, which accounts for his or her thermal fragility and want for storage and cargo in freezers at numerous temperatures.

It’s of appreciable scientific and public curiosity to know the immunogenicity of the main vaccines in absolute and, to the extent doable, comparative phrases. Right here, we have now reviewed antibody and T cell immune response information derived from printed research of vaccines that had been examined in nonhuman primates (NHPs) after which progressed into human section 1/2 trials or which are in human trials and not using a prior NHP experiment (Tables 2 to 4). Now we have additionally evaluated macaque vaccine problem experiments, together with how they had been carried out, because the outcomes are related to understanding the protecting potential of SARS-CoV-2 vaccines (Desk 3). The NHP experiments are described in (1–13), and the human trials are described in (14–32).

The Chinese language authorities licensed the CanSinoBIO Ad5-nCoV vaccine to be used on army personnel in June 2020, presumably on the premise of the section 1/2 trial information (23, 24). In August 2020, the Russian authorities authorized an Ad5 and Ad26 adenovirus vector vaccine, Gam-COVID-Vac (additionally known as Sputnik V), after minimal security testing and with no proof of protecting efficacy, a number of weeks earlier than section 1/2 trial information had been printed (28). Reservations have been expressed concerning the suitability of Ad5 vaccines to be used in areas of excessive HIV-1 incidence, primarily based on the dangers of elevated HIV-1 acquisition (44). This concern would apply to each the CanSinoBIO Ad5-nCoV and the Gam-COVID-Vac vaccines (23, 24, 28). The approval processes that shall be utilized to the vaccines of the U.S. authorities–supported OWS program are outlined in short elsewhere (33).

The immunogenicity of a few of the >150 vaccine candidates now in preclinical growth worldwide has been examined in small animals and, in some instances, NHPs. These reviews are past the scope of this assessment, though we and others have summarized a number of beforehand (34, 35, 45). Small animal immunogenicity research that straight relate to the vaccine candidates we assessment listed here are described in a number of of the papers on NHP experiments and human trials and likewise in (42, 46–50).

The primary indications of vaccine efficacy emerged throughout November, 2020, with the looks of seven press releases. “Science by press release” is a removed from best mechanism for the dissemination of necessary information however is comprehensible within the context of the COVID-19 pandemic. The data within the press releases was typically fairly restricted, and, after all, the info weren’t peer reviewed. In some instances, extra questions had been raised than solutions given. Nonetheless, it was clear that important and significant ranges of efficacy had been being completed. Not one of the press releases reported extreme security issues. We summarize what’s now recognized close to the tip of this assessment.



Antibodies induced by the S protein–primarily based immunogens are typically measured in two methods. Immunoassays, often however not all the time enzyme-linked immunosorbent assays (ELISAs), quantify antibody binding to the S protein or fragments thereof, such because the RBD. Neutralization assays assess the skills of NAbs to inhibit SARS-CoV-2 an infection of goal cells (51, 52). The binding and NAb assays each have worth, and titers derived from them typically correlate moderately properly. Nevertheless, neutralization assays quantify antibodies that block an infection, whereas ELISAs and different binding antibody assays additionally detect antibodies that lack these properties (non-NAbs) (Figs. 1 to three). Different assays are generally used, for instance, to detect antibodies that inhibit the binding of the S protein or its RBD to a soluble type of the angiotensin-converting enzyme 2 (ACE2), which is the entry receptor for SARS-CoV-2. We limit our dialogue to binding antibodies and NAbs, with some exceptions. A repeated prevalence within the papers we summarize is the usage of COVID-19 convalescent sera or plasma as comparators for vaccine-induced antibody responses. Now we have ignored all of those datasets. The serum/plasma panels differ among the many numerous research, and the vary of antibody titers seen in COVID-19 sufferers can span a 5-log vary and range significantly additionally throughout convalescence (34, 35, 51, 53–55). Accordingly, we have now not discovered the convalescent serum panels useful when gauging the relative immunogenicity of the assorted vaccine candidates. There’s a compelling must now assemble and use a regular panel containing neutralizing MAbs and/or validated convalescent plasma or purified immunoglobulin G (IgG) preparations for these comparisons (34, 35, 51).

As we have now famous beforehand, totally different analysis teams use totally different assays and measure antibody binding and virus neutralization in a different way, which enormously complicates comparisons of datasets (34). The totally different measurements made within the authentic papers are defined in Desk 5 and Figs. 1 to three. How binding and NAb titer and safety information derived from animal experiments that use totally different infectious doses of problem virus might be associated to one another has been modeled and mentioned elsewhere (51, 52, 56). The vary of SARS-CoV-2 vaccine–induced antibody titers seen in teams of NHPs and people typically exceeds 100-fold and might be as excessive as 1000-fold. As famous beforehand, the existence of such a variety of responses has implications for the proportion of a inhabitants {that a} vaccine can shield (34). The titer spreads reported within the numerous major papers are value evaluating from this angle.

Antibody responses within the numerous research are often measured for under a brief interval after the ultimate immunization or, within the case of some macaque experiments, after the virus problem. The identical constraint applies additionally to the T cell information. In a single examine the place evaluations had been carried out for longer than is regular, rhesus macaques had been immunized twice with the Pfizer/BioNTech BNT162b2 mRNA vaccine. The height anti–S1 protein antibody titers then declined ~5-fold over a 28-day interval, whereas NAb replicating virus (RV) median inhibitory dilution (ID50) titers additionally dropped ~5-fold within the high-dose (100 μg) group (11). We estimate that the early-phase half-life of those antibody titers is only one to 2 weeks. The lack of know-how of the longevity of SARS-CoV-2 vaccine–induced immune responses in people is a considerable hole that may want filling.

In nearly all the papers we assessment, antibody responses are measured solely in serum. There was little or no consideration paid, so far, to mucosal immune responses, which appears unlucky given how SARS-CoV-2 is transmitted and the place it predominantly replicates. Accordingly, we can’t deal with mucosal immunity on this assessment, aside from by noting that one latest preclinical examine of a chimpanzee adenovirus vaccine (totally different from the AstraZeneca scientific candidate) in mice highlights how necessary inducing and characterizing mucosal immune responses would possibly develop into (50).

Briefly, it’s usually tough to examine two totally different papers on vaccines A and B and conclude with certainty that one induces the superior immune response. Data of how vaccines of various designs typically carry out will help type judgments, however there should all the time be caveats.



T cell responses to vaccine immunogens are typically measured by quantifying the quantity of cytokine expressed by a T cell after particular antigenic stimulation from a peptide, protein, or vector-delivered antigen. The enzyme-linked immune absorbent spot (ELISpot) assay is most frequently used, or variants thereof, peripheral blood mononuclear cells (PBMCs) being the most common supply for T cells. Interferon-γ (IFN-γ) secretion is probably the most generally chosen cytokine output, however different cytokines are generally additionally measured, as is the manufacturing of granzyme B. Cytokine stream cytometry (CFC) is commonly used as a readout, and there may be typically an excellent correlation between ELISpot and CFC assay outcomes. A bonus of the CFC assay is that it could actually straight establish the phenotype of responding T cells, which requires depletions of mobile subsets in ELISpot assays. Assays for antigen-specific CD4+ T cells generally measure the up-regulation of activation-induced floor markers. Nevertheless, these strategies don’t measure T cell avidity or check the efficiency of cells in viral inhibition assays (57). Right here, we confine our dialogue to ELISpot assays, with some exceptions.

Relying on the vaccine candidate antigen, a T cell assay can use particular person peptides, principally derived from the S protein, pooled or matrix-pooled peptides, or protein or vector-expressed antigen as a supply of peptides to bind to the main histocompatibility complicated molecules which are expressed on the cell floor and acknowledged by a selected T cell receptor. As cross-reactive T cells are recognized to happen, most assays won’t particularly establish a response that was elicited by prior publicity to a cross-reactive pathogen (or a special vaccine). For instance, an earlier an infection with one of many frequent chilly coronaviruses would possibly result in a secondary reminiscence response that might skew the end result of the SARS-CoV-2 vaccine trial evaluation, until prior an infection by these different coronaviruses is an exclusion criterion (which is never if ever the case).

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ELISpot outcomes are often expressed as spot-forming cells (SFCs) per 106 enter PBMCs, however this isn’t a uniform follow. For instance, some investigators use SFCs per 105 cells as their learn out; we multiply their values by 10 and report them as SFCs per 106 cells. We additionally use the abbreviation SFC moderately than SFU (spot-forming models) when the latter is used within the authentic paper. There are additionally variations in methodologies, together with the size of time between blood draw and cryopreservation, the strategy used for thawing, the peptide focus used, the period of peptide incubation with the cells, the time taken to finish the assay, and whether or not responding T cells are separated. All of those components can have an effect on an ELISpot end result and have to be thought of when evaluating totally different research. A normal function of the papers we have now summarized is an absence of element on how the assays had been carried out. It could even be helpful if photographs of key ELISpot plates had been supplied as uncooked information, to permit the spots to be recounted. The timing of when cell samples are collected after a vaccine prime or enhance can also be related. Thus, the time-dependent decay of circulating T cells impacts the magnitude of the responses measured in vitro, to a higher extent than applies to the extra steady antibody responses.

Considerations have been expressed that SARS-CoV-2 vaccines might exacerbate illness in contaminated animals, primarily based on information arising in earlier animal mannequin experiments with vaccines in opposition to different coronaviruses (34, 35, 45, 58–60). One specific potential downside is known as vaccine-associated enhanced respiratory illness (VAERD) (59). Whereas it’s not doable to find out whether or not VAERD shall be an issue with SARS-CoV-2 vaccines earlier than the end result of efficacy trials and after licensure security assessments, the pulmonary dysfunctions are related to elevated manufacturing of interleukin-4 (IL-4), IL-5, and IL-13, eosinophil recruitment, and impeded CD8+ T cell responses (59–61). This sample of immune responses is indicative of T helper 2 (TH2) polarization. Accordingly, a few of the NHP and human experiments embrace analyses of in vitro cytokine launch profiles, to hunt indicators of undesirable, TH2-biased responses. Thus far, TH2 responses have hardly ever been seen. We briefly notice the outcomes of those analyses after they had been carried out.



Immunogenicity research have been carried out in rhesus or cynomolgus macaques or, in a single case, baboons (1–13). The immunogens had been typically examined beforehand in small animals, usually however not all the time mice, to supply preliminary assessments of their efficiency and to supply some indication of the dose or dose vary to then consider in NHPs. Right here, we focus solely on the NHP research themselves; the first papers needs to be consulted for the small-animal information. On the whole, the NHP experiments additionally concerned security assessments. The outcomes had been unexceptional in that no important issues had been reported within the major papers, which ought to, once more, be consulted for particulars. Key serum antibody titer values recorded on this part are summarized in Desk 2 and, for information obtained on the time closest to problem, additionally in Desk 3. T cell response information are equally summarized and tabulated, though these assays weren’t carried out in a number of of the research. In all instances, the vaccines had been administered intramuscularly, which additionally applies to the human scientific research (see under). Nevertheless, small-animal research of a chimp adenovirus vaccine and an Ad5 vaccine (not the AstraZeneca/Oxford and CanSinoBIO scientific candidates, respectively) recommend that viral vectors is likely to be very fruitfully delivered by the intranasal route as an alternative (50, 62).

Often, a number of subgroups of macaques had been rolled over right into a SARS-CoV-2 problem examine, or the optimum routine was examined in a de novo experiment. Some particulars and the outcomes of the virus challenges are summarized individually under and in Desk 3.

The primary macaque immunogenicity paper to look described PiCoVacc, the Sinovac β-propiolactone–inactivated, Vero cell–produced virus vaccine (1). Be aware that this vaccine was renamed CoronaVac for human scientific trials (Desk 1) (14). Two vaccine doses (3 and 6 μg of viral protein) with an Alum adjuvant had been examined on teams of 4 rhesus macaques by three immunizations on days 0, 7, and 14. The 6-μg dose elicited barely the stronger antibody responses on day 21, when the anti–S protein geometric imply (GM) ELISA end-point titers had been ~12,800 and NAb GM median inhibitory dilution (ID50) values had been ~50 in an RV assay (Desk 2). Antibodies particular for the RBD dominated the antibody response to the inactivated virus vaccine, which is related to understanding the end result of the problem experiment (see under).

The Sinopharm/Beijing Institute of Organic Merchandise (BIBP) inactivated virus vaccine, BBIBP-CorV, was additionally produced in Vero cells and inactivated with β-propiolactone. Combined with Alum adjuvant, three totally different doses (2, 4, and eight μg of viral protein) had been administered to teams of 10 cynomolgus macaques on days 0, 7, and 14 (2). The ensuing NAb titers, measured in an RV assay, had been dose dependent, with a GM ID50 worth of ~210 reported for the highest-dose group on day 21 (Desk 2).

The ChAdOx1 nCoV-19 recombinant virus vector expresses a nonstabilized type of the SARS-CoV-2 S protein (3). Teams of six rhesus macaques acquired this vaccine (2.5 × 1010 particles) both as soon as (day 0) or twice (days 0 and 28) in a prime-boost protocol. Within the single-dose group, the anti–S protein median end-point titer on day 14 was ~600, and the median NAb ID50 worth was ~20 in an RV assay. The second dose boosted these responses to ~28,000 and ~280, respectively, on day 42 (Desk 2). The animals had been challenged with SARS-CoV-2 on day 28 (one-dose group) and day 56 (prime-boost group), as summarized under (Desk 3).

Rhesus macaques had been used to establish and consider the optimum design of the Janssen Ad26.COV2.S vaccine candidate (4). First, antibody responses to seven totally different S protein variants had been in contrast utilizing a spread of assays, resulting in the choice of the optimum Ad26 S.PP design. After a single dose of this immunogen, median RBD-ELISA end-point titers at week 4 had been ~4000, whereas the pseudo-virus (PV) and RV NAb median ID50 values had been 408 and 113, respectively (Desk 2). In an IFN-γ ELISpot, at week 4, the median response elicited by the S.PP vaccine was solely ~80 SFCs per 106 cells. The information had been inadequate to confidently assess the TH1 versus TH2 bias, as solely IFN-γ and IL-4 responses had been measured. T cell response information had been presumably not factored into the choice to decide on the S.PP assemble because the scientific candidate, as this virus was the least immunogenic of the seven variants from the attitude of inducing CD4+ and CD8+ T cell immunity. The antibody responses had been prioritized (4). In a bigger and extra complicated examine, numerous Ad26.COV2.S vaccine-dosing parameters had been evaluated in grownup macaques, together with the variety of virus particles administered (5 × 1010 versus 1 × 1011), the advantages of 1 dose versus two doses, and the interval between the primary and second dose in a two-dose routine (4 weeks versus 8 weeks) (5). Antibody and T cell responses had been assessed utilizing comparable assays to the preliminary examine (Desk 2). The varied intergroup comparisons confirmed that two doses of 5 × 1010 virus particles given at weeks 0 and eight had been the superior routine for human research (5). These findings presumably influenced the choice to start a section 3 trial of the two-dose routine, to complement the continuing one-dose trial (see under). An extra facet of the macaque experiment was testing in aged macaques, outlined as animals 14 to 22 years outdated. Right here, the immunogenicity of the popular two-dose, 0- and 8-week routine was discovered to be akin to what was seen within the youthful grownup animals (5). One remaining aspect of the general experiment was the usage of an Alum-adjuvanted recombinant, stabilized S protein, principally for assessing TH1 versus TH2 bias in comparison with the Ad26 vector regimens. As anticipated, the responses to the Ad26 virus vectors had been extra TH1 polarized than these to the Alum-adjuvanted S protein (5). No virus challenges had been performed.

DNA vaccines expressing six totally different SARS-CoV-2 S protein variants, together with the full-length S protein and the RBD, had been examined, with out adjuvant, in rhesus macaques (6). Median end-point anti–S protein titers at week 5 different reasonably with the immunogen however had been 140 to 180 for the full-length S protein and RBD immunogen teams. Midpoint NAb titers at week 5 additionally different by immunogen, with median ID50 values of ~50 to 200 and ~30 to 40 in PV and RV assays, respectively (Desk 2). The complete-length S protein assemble elicited considerably stronger NAb responses than its RBD counterpart. At week 5, T cell responses had been detectable in ELISpot assays with pooled S peptides (~80 SFCs per 106 cells within the S group; Desk 2). Intracellular staining confirmed IFN-γ responses each in CD4+ and CD8+ subpopulations; the responses to full-length S had been stronger than to S1 and RBD. Final, IL-4 responses had been barely detectable, which is appropriate with a TH1 bias of the mobile immune responses (6).

INOVIO’s INO-4800 S protein–primarily based DNA vaccine was given to 5 rhesus macaques in 1 mg of doses at weeks 0 and 4 by an intramuscular electroporation gadget that gives a light electrical shock to open membrane channels in muscle cells (7). The height anti–S protein GM end-point titer (week 6) was ~130,000 but it surely dropped ~40-fold by the point of problem at week 17. The binding antibody end-point titers in opposition to the RBD had been ~5-fold decrease than in opposition to the S protein at week 6. Within the PV NAb assay, the height GM ID50 titers had been ~1000 however had declined to ~250 by week 12 (i.e., a four- or fivefold titer lower over 6 weeks in opposition to the 2 PVs examined). Low titers of anti S protein IgG (~10) had been additionally detected in bronchoalveaolar lavage (BAL) samples from vaccinated animals. An IFN-γ ELISpot was used to measure T cell responses triggered by 5 peptide swimming pools at week 6. Alerts had been seen with PBMCs from 4 of the 5 animals, with a spread of 0 to 518 SFCs per 106 cells and an arithmetic imply (AM) worth of ~140. By week 12, the imply worth had declined to solely ~30 (7).

The Moderna S protein–primarily based vaccine candidate, designated mRNA-1273, was additionally examined in rhesus macaques (8) The lipid-encapsulated mRNA formulation was given intramuscularly at doses of 10 or 100 μg to every of two teams of eight animals, and at weeks 0 and 4, anti–S protein ELISA information had been offered solely within the type of space underneath the curve (AUC) values, precluding direct comparability with different research. For the high-dose group at 4 weeks after the second dose, the NAb GM ID50 values in PV and RV assays had been 1862 and 3481, respectively. In a CFC assay, TH1 responses had been dose-dependent, whereas TH2 and CD8+ T cell responses had been at most minimal. Though all animals responded based on prespecified standards, the T cell assay indicators had been typically weak, even on the highest vaccine dose. There have been no variations in TH1- or TH2-associated cytokines or chemokines in BAL samples from the vaccine and management animals (8).

A paper describing the immunogenicity of the Pfizer/BioNTech BNT162b2 S protein–expressing mRNA vaccine in mice and rhesus macaques appeared a number of weeks after one which reported on the efficiency of the identical vaccine in people (Tables 2 to 4) (11, 25). Both 30- or 100-μg doses of the mRNA got intramuscularly to the macaques on days 0 and 21. Serum anti–S1 antibodies had been quantified by ELISA on days 21, 28, 35, 42, and 56 and offered as ELISA models per milliliter (derived from comparability with a regular curve). On day 28, these values had been 30,339 and 34,668 for the 30- and 100-μg teams, respectively, however had declined ~5- to 7-fold to 4236 and 6317 by day 56. The sample of NAb information, assessed by an RV assay, was comparable; the height NAb GM ID50 values had been 962 on day 35 and 1689 on day 28 for the 2 dosing teams, however by day 56, that they had dropped 3- to 5-fold to 285 and 310, respectively. The inferred half-life for the early-phase decline is roughly 1 week for the binding antibodies (ELISA) and a couple of weeks for NAbs, though the latter titers declined extra quickly on the later time factors. Antibody decay charges this excessive are a possible concern for the longevity of any safety seen in people, notably if they’re additionally seen in trials of the opposite vaccines. T cell responses, measured by IFN-γ ELISpot on days 28 and 42, had been ~750 SFCs per 106 PBMCs for each dosing teams, with IL-4 responses under 250 SFCs per 106 PBMCs (11).

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A 3rd S protein–primarily based mRNA vaccine has now been described, MRT5500 from Sanofi Pasteur (13). Preliminary research in mice led to the selection of a full-length S protein assemble, 2P/GSAS, that comprises the generally used two proline mutations (2P) and a furin cleavage website knockout mutation (GSAS). The 2P/GSAS mRNA, formulated as lipid nanoparticles, was examined in a cynomolgus macaque dose-ranging examine. Thus, 15, 45, or 135 μg of the mRNA got on days 0 and 28, resulting in NAb responses on day 35 that trended upward in a dose-dependent method. For the 135-μg dose group, the NAb GM ID50 titers had been 2871 (PV assay) and 1877 (RV assay). PBMC T cell responses, assessed utilizing a cytokine launch (ELISpot) assay, had been TH1-biased (IFN-γ launch however not IL-13). The magnitude of the macaque IFN-γ response was very low, round 30 to 40 SFCs per 106 PBMCs (Desk 2). No virus problem was carried out (13).

The Novavax NVX-CoV3273 vaccine is an insect cell–derived S protein that’s blended with detergent to type what are described as nanoparticles (9, 10, 21, 63). That formulation is mixed with the Matrix-M adjuvant. The immunogenicity examine in baboons in contrast 1-, 5-, and 25-μg doses of the S protein in a two-dose (days 0 and 21) routine, whereas a fourth group acquired 25 μg with no adjuvant. Antibody assays on days 21, 28, and 35 confirmed that the optimum dose was 5 μg with adjuvant, with the height response reached by day 28. The best GM anti–S protein GM median efficient dilution (EC50) and NAb ID>99 values had been 174,000 and 17,000, respectively (9). The identical protein/adjuvant mixture was then examined in cynomolgus macaques (10). The animals had been immunized on days 0 and 21 at totally different doses, with each the protein and adjuvant quantity various (protein at 2.5-, 5-, and 25-μg doses). On the highest dose (25 μg of S protein, 50 μg of adjuvant, mirroring one of many human examine teams; see under), the anti–S protein GM ED50 worth on day 35 was 469,739. Be aware that binding antibody information had been offered as ED50 values, not the extra traditional end-point titers, which might be considerably larger (maybe 10- to 100-fold). Within the RV NAb assay, the CPE100 (inhibition of ~100% of the cytopathic impact, roughly equal to ID>99) was measured, with GM values starting from 17,920 to 23,040 within the totally different dosing teams. It needs to be famous that the neutralization titers for close to full efficacy of neutralization measured in these research (CPE100 or ID>99) shall be considerably decrease than the extra typical ID50 values, though we can’t estimate by how a lot. In abstract, the NAb titers in these papers are primarily based on extremely stringent assessments of virus neutralization, which needs to be borne in thoughts to keep away from underestimating the sturdy antibody immunogenicity of NVX-CoV3273 (9, 10).

One other recombinant S protein vaccine has been examined in rhesus macaques, on this case, from the corporate, Clover Biopharmaceuticals (12). The S protein comprises a C-terminal “TRIMER-Tag,” to advertise trimer formation and stability, and was produced in a high-yielding, steady Chinese language hamster ovary cell line. After pilot experiments in mice, rhesus macaques got 30 μg of the S protein on days 0 and 21 in both AS03 or CpG/Alum adjuvant, or car as a management (Desk 2). On day 35, the strongest anti–S protein antibody responses had been within the AS03 group (GM end-oint titers of ~17,497 versus ~3157 within the CpG/Alum group). On the identical time, the GM NAb ID50 titers within the AS03 group had been 5227 and 20,234 in a PV and an RV assay, respectively. The D614G mutation within the recombinant trimer didn’t have an effect on ACE2 binding or the competitors therewith by murine immune sera. The partial safety noticed in a virus problem experiment is summarized under (Desk 3). A section 1 scientific trial of the S protein with both AS03 or CpG/Alum adjuvant is now in progress in China (12).



When deciphering the end result of macaque problem experiments, it needs to be borne in thoughts that SARS-CoV-2 doesn’t trigger a deadly COVID-19–like illness in these animals. The macaques do turn out to be in poor health, rhesus maybe extra so than cynomolgus, however the illness course is usually gentle, self-limiting, and overcome inside ~2 weeks (51, 64–66). On the whole, the assorted vaccines cut back the severity of this gentle illness, together with by decreasing and even stopping the transient lung injury that may be seen in postmortem samples taken from management animals. No indicators of vaccine-mediated enhancement of an infection, together with VAERD, had been reported. The commonest approach during which vaccine efficacy is assessed is by figuring out the viral load (VL) in samples from numerous locales and tissues at quick intervals in the course of the week after problem. In some experiments, each viral RNA copies per milliliter and subgenomic RNA copies per milliliter are decided, the latter avoiding issues related to the presence of residual problem virus and extra unambiguously demonstrating viral replication within the contaminated animal (Desk 3) (6, 51, 67). Lung pathology was additionally typically assessed, though the factors chosen are inclined to range among the many totally different experiments. For a very detailed dialogue of the variables related to problem experiments in macaques and the way they’re finest interpreted, see (51).

Antibody titers within the animals on or very near the day of problem had been reported in a few of the papers and are summarized under and likewise in Desk 3. Within the different papers, the antibody information had been derived at an earlier time level (Desk 3). The inconsistencies in how the totally different research had been performed and/or reported are one other issue that blurs makes an attempt to match and interpret the performances of the totally different vaccines. Solely 4 of the reviews embrace information on T cell responses at any time level earlier than problem, which limits understanding of any position they might play (Desk 2) (4–7). In a separate part, we talk about what, if any, correlates of safety (CoPs) might be inferred from a few of the problem experiments.

A lot of the experiments concerned SARS-CoV-2 challenges inside a number of weeks of the ultimate (or solely) vaccine dose, i.e., at a time when the immune response is prone to be near its peak. The exception is the INOVIO DNA vaccine examine during which the problem was delayed by 13 weeks (7). The subsequent longest delays are 55 days after the second dose of the BNT162b2 mRNA vaccine and 42 days after the supply of a single dose of the Ad26.COV2.S vaccine (Desk 3) (4, 11). Thus, it’s not but recognized whether or not these numerous vaccines could be as efficient in opposition to challenges performed many months after the immunization protocol was accomplished. Extrapolating to what would possibly occur when vaccinated people turn out to be uncovered to SARS-CoV-2 over the following months or years will not be doable.

One other subject when contemplating these macaque experiments issues the SARS-CoV-2 problem itself. There isn’t a typically accepted commonplace, and numerous totally different problem virus shares (in a number of instances, of unspecified origin) had been used. The problem dose additionally varies 100-fold, and the route of problem is one other variable (Desk 3). In a single experiment, the virus was even administered by 4 totally different routes (3). All of those protocol variations represent yet one more issue hindering cross-study comparisons (34, 51). As a normal precept, it is going to be simpler to guard in opposition to a low dose of a problem virus than a better one, all different issues being equal. Thus, would a vaccine that protected in opposition to a comparatively low problem dose be as protecting in opposition to the 100-fold larger dose utilized in different experiments (34, 51)? Or would its safety break down underneath these situations? We return thus far on the finish of this part. Problem doses for vaccine experiments are historically predetermined in naïve animals, to establish an inoculum measurement that’s neither too low to be persistently infectious nor too excessive to guard in opposition to. It’s hardly ever clear from the papers whether or not these titrations had been carried out. In a single report, nasal swab VL samples taken from SARS-CoV-2–contaminated people and from the virus-challenged macaques quickly after an infection had been stated to be comparable (~1 × 106 RNA copies per milliliter) (8). Nevertheless, the preliminary an infection and subsequent replication efficiencies are prone to differ considerably between the 2 species so it’s not clear that this comparability is significant.

Within the Sinovac PiCoVacc examine, teams of 4 rhesus macaques had been immunized with both 3 or 5 μg of the inactivated virus vaccine on days 0, 7, and 14 and challenged intratracheally with the CN1 pressure of SARS-CoV-2 on day 22 (1). Right now, the anti–S protein GM end-point titer was ~12,800, whereas the NAb GM ID50 titer was ~50 when measured 7 days earlier. All of the vaccinated animals grew to become contaminated after problem, however illness severity was decreased in comparison with the management group (adjuvant solely) as judged by lung pathology assessments. VLs (i.e., viral RNA) had been continuously detected at excessive ranges in lung samples from management animals however in not one of the high-dose vaccine recipients and solely sporadically at considerably decrease ranges within the low-dose group. Viral RNA ranges in throat swabs had been additionally decrease and declined extra quickly, notably within the larger vaccine-dose group (Desk 3). The noticed will increase in NAb titers day 7 after an infection could also be per an anamnestic antibody response (1).

The Sinopharm/BIBP inactivated virus vaccine experiment concerned two teams of 4 cynomolgus macaques that had been immunized with totally different doses (2 or 8 μg of viral protein) on days 0 and 14 (2). Binding antibodies weren’t measured. The NAb GM ID50 values in an RV assay had been ~200 and ~230 within the low- and high-dose teams when the animals had been challenged on day 24 with a SARS-CoV-2 isolate from the Chinese language Heart for Illness Management and Prevention, by the tracheal route (Desk 3). There have been no modifications in physique temperature in both the vaccine or placebo teams over the following 7 days, which is indicative of a light illness course. Viral RNA in all lung lobes was analyzed postmortem, however none was detected in any lobe taken from vaccine recipients (in both dosing group). In distinction, the RNA copy quantity per milliliter ranged from ~30,000 to three,000,000 within the decrease lobes of the management animals. Lung pathology was additionally prevented or decreased within the vaccine teams. Though viral RNA in throat swabs grew to become undetectable 7 days after problem within the high-dose group, different proof means that these animals did turn out to be contaminated, albeit to a a lot lesser extent than the management and low-dose vaccine teams. Thus, gastrointestinal virus (detected in anal swabs) remained steady within the excessive dose at ~100 RNA copies per milliliter from days 3 to 7, whereas the corresponding values within the two different teams fluctuated round 100,000. Whereas this examine solely analyzed viral, not subgenomic, RNA, it appears extremely unlikely that gastrointestinal viral RNA might merely signify residual problem virus. Thus, the higher-dose animals had been no less than strongly, however apparently not utterly, protected against an infection, and in each dosing teams, the vaccine decreased the extent of virus replication after an infection (2).

All 12 of the ChAdOx1-vaccinated macaques grew to become contaminated after they had been challenged 28 days after their remaining immunization (they acquired both one or two vaccine doses; see above) (3). The SARS-CoV-2 problem pressure was WA1-2020 (MN985325.1). On the time of problem, the median binding antibody end-point titers had been ~6300 and median NAb ID50 values ~60 in an RV assay (Desk 3). The vaccinated animals had fewer signs than the management group, much less lung injury, and decrease VLs (measurements included subgenomic RNA) in BAL and lung samples. No virus was detected in BAL samples from the vaccinated animals on day 5, however subgenomic RNA could possibly be detected in lung samples from some animals in each teams. No antibody or T cell information after problem had been reported, so it’s unknown whether or not there have been anamnestic responses to the infecting virus (3).

Seven totally different Ad26-based vectors got as soon as to teams of 4 to 6 rhesus macaques earlier than problem 6 weeks later with an unspecified isolate of SARS-CoV-2 (4). In comparison with a management group of 20 animals, VLs in BAL and nasal swabs had been considerably decreased in every of the seven Ad26 vector teams, by >5 logs within the case of BAL samples. The perfect performing vector, from this angle, was the one designated S.PP; it was chosen to turn out to be the Ad26.COV2.S scientific vaccine candidate. General, the authors assessed that 17 of the 32 vaccinated macaques had been protected against an infection, judged by the VL information. There was no proof for anamnestic B and T cell responses within the protected Ad26.COV2.S-vaccinated animals, though NAb titer will increase had been seen in different vaccine teams. The strongest, and maybe full, safety was seen within the S.PP group. Thus, virus (subgenomic RNA) couldn’t be detected in BAL from 6/6 and in IN swabs (INSs) from 5/6 animals (4). Extra just lately, hamsters immunized as soon as with the S.PP-expressing Ad26 scientific vaccine candidate had been protected against extreme illness when challenged with SARS-CoV-2 nasally 4 weeks later (49).

In one other examine, rhesus macaques had been immunized intramuscularly with S protein–expressing DNA plasmids at weeks 0 and three and challenged at week 6 with an unspecified SARS-CoV-2 isolate (6). The entire 10 management animals grew to become contaminated, buying BAL and nasal swab peak subgenomic RNA copy ranges within the vary 104 to 107 per milliliter. Nevertheless, 8 of the 25 vaccine recipients had been RNA detrimental in BAL and nasal swab samples, whereas median subgenomic RNA ranges within the different 17 macaques had been 3 to 4 logs decrease than the median values from the ten management animals. Even when subgenomic RNA was undetectable in vaccinated animals, the commentary of anamnestic antibody and T cell responses does suggest that the animals weren’t utterly protected against an infection. As an alternative, initially replicating virus might have been suppressed by vaccine-mediated immunity (6).

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The INOVIO INO-4800 DNA vaccine was given to 5 rhesus macaques at weeks 0 and 4 (7). The SARS-CoV-2 problem (USA-WA1/2020 pressure) was then delayed till week 17 (i.e., 13 weeks after the second immunization), a considerably longer interval than applies within the different research summarized right here. Upon problem, all of the macaques grew to become contaminated, judged by VLs in numerous samples. Nevertheless, VLs within the vaccinated group had been decrease and declined extra rapidly than in 5 management animals, the discount in medians being within the 10- to 300-fold vary relying on the pattern website and the time level assayed; the distinction was important for BAL however not INS samples. Antibody and T cell recall responses had been quantified within the animals after virus problem. Thus, by 14 days after problem, anti–S protein antibody and NAb measurements had been larger (~10- to 30-fold) within the vaccinated than management animals, whereas there was an ~2-fold improve in IFN-γ indicators. General, the vaccine-mediated discount in viremia after problem was attributed to recall responses (i.e., T and B cell reminiscence) (7).

Two dosing teams of eight rhesus macaques had been immunized with the Moderna mRNA-1273 vaccine at weeks 0 and 4 after which challenged with the USA-WA1/2020 pressure of SARS-CoV-2 at week 8 (8). Judged by VLs, most (~7 of 8) of the animals within the higher-dose group had been protected, however most (~5 of 8) of the lower-dose group grew to become contaminated (the precise numbers range per time level and rely on the VL pattern website). There have been indications of anamnestic responses in some animals, together with in BAL. Postmortem analyses of the lungs discovered little or no indicators of irritation within the higher-dose group, however some indications of pathology within the lower-dose animals that grew to become contaminated. Neutralization titers in each PV and RV assays correlated inversely with INS VLs; virus-specific IgG and IgA ranges in BAL had been elevated within the high-dose group on days 2 to 7 after problem, an anamnestic response that will maybe have contributed to VL discount. Within the high-dose, high-protection group, the GM ID50 values from PV or RV NAb assays had been >900 in 7/8 animals, whereas the corresponding values had been <900 for 7/8 animals within the low-dose, low-protection group. The information sample implied that NAbs had been protecting within the high-dose group (8). Within the BNT162b2 vaccine examine, six rhesus macaques immunized on days 0 and 21 with 100 μg of the mRNA and three naïve controls had been challenged on day 76 general with 1.05 × 106 plaque-forming models (PFU) of the USA-WA1/2020 pressure of SARS-CoV-2 by the intranasal (IN)–intratracheal (IT) routes (11). Antibody titers on the day of problem weren't reported, however measurements made on day 56 are summarized above and in Tables 2 and three. An infection was monitored by viral RNA copies in BAL, nasal swabs, and oropharyngeal swab on days 3, 6, and 10. No viral RNA was detected at any time within the BAL samples from the immunized macaques however was current in management samples. The same information sample, after day 1, was seen when the nasal swabs had been analyzed, and within the oropharyngeal swabs, 2/6 had been optimistic on day 3. The GM reductions in VL measured as viral RNA copies had been ~3.0 log for BAL, 1.5 log for INSs, and a couple of.5 log for oropharyngeal swabs (the primary and final of which had been statistically important). One uncommon facet of the experiment was that the entire absence of illness signs within the management animals (and, after all, additionally within the vaccine recipients). As famous above, SARS-CoV-2 an infection typically causes average illness in rhesus macaques, together with in different vaccine experiments. Why no such signs had been seen within the current examine was not defined however might maybe be rooted within the problem virus inventory or the origins of the macaques (11). The adjuvanted Novavax NVX-CoV2373 recombinant protein vaccine was given to cynomolgus macaques on days 0 and 21 earlier than the animals had been challenged with the WA1 pressure of SARS-CoV-2 through the nasal and tracheal routes on day 35 (10). Judged by VLs (subgenomic RNA) in BAL and nasal swabs, carried out 2 and 4 days later, each animal was virus-negative apart from one within the lowest-dose group that had a weakly optimistic BAL pattern. Postmortem lung samples within the vaccine teams confirmed no signal of the pathologies that had been seen within the management animals. To the extent that may be judged, most vaccinated animals might have been utterly protected against an infection. This final result might replicate the very excessive GM antibody titers on the day of problem (anti–S protein ED50 of 469,739 and NAb CPE100 of 23,040) (10). Clover Biopharmaceuticals examined their recombinant TRIMER-Tag S protein and AS03 or CpG/Alum adjuvants in rhesus macaques (12). After two vaccine doses on days 0 and 21, the animals had been challenged on day 35 with 2.6 × 106 median tissue tradition infectious dose (TCID50) of SARS-CoV-2 by the IN and IT routes (Desk 3). Each of the trimer/adjuvant teams of animals had been partly protected as measured by physique mass and temperature and VL in anal, throat, and tracheal samples however much less so in nasal swab samples. Nevertheless, VLs within the lungs indicated full safety in each of the trimer teams, in contrast with the car controls. Serum Ab responses dropped a bit of within the week after problem, which the authors urged mirrored the formation of immune complexes with the incoming virus and therefore Ab-mediated virus clearance (12). Whether or not this clarification is right stays to be decided. In abstract, all the vaccines examined so far have conferred a considerable diploma of safety to the immunized macaques. In some macaques, there may be affordable proof for full safety (i.e., “sterilizing immunity”), however the extra frequent final result is a discount within the severity of the already gentle illness course seen in management animals. We talk about within the subsequent part what immune components and different variables might have influenced the outcomes of the totally different experiments. In respect of what the outcomes might imply for vaccine efficacy in people, we notice that it's typically simpler to guard animals in opposition to gentle infections than extreme ones. Therefore, it's onerous to evaluate whether or not and the way any of the current findings in macaques would possibly translate to the subset of people who want safety from extreme and deadly COVID-19. Furthermore, as famous above, it's not recognized whether or not the assorted vaccines would nonetheless shield macaques, and by extrapolation people, after a considerable interval (a number of months) has elapsed.  


It’s notable that within the numerous macaque immunization research, comparable outcomes of virus challenges had been related to substantial (~2000-fold) variations in serum antibody titers to the S protein, the recombinant protein vaccines being the strongest immunogen for inducing binding antibodies (9, 10) or NAbs (9, 10, 12) (Desk 2). It might additionally moderately be concluded that the ChAdOx1 vaccine will not be a powerful inducer of antibody responses to the S protein, notably when given solely as soon as (Desk 2) (3). The identical conclusion might be made concerning the DNA plasmid vaccines (Desk 2) (6, 7). Are the serum antibody responses induced by the weaker vaccines solely accountable for any safety that was conferred? Maybe, mobile immune responses or another unmeasured issue, reminiscent of mucosal IgA, had been contributory? The potential protecting position of mucosal immunity is highlighted by the outcomes of experiments involving a ChAd virus vector in mice (50). These observations are just like these in research of different vaccines, reminiscent of HIV-1 Env, the place solely protein-based immunogens induce very sturdy antibody titers (68).

What protected the vaccinated animals from SARS-CoV-2 an infection and/or illness? CoPs (correlates of safety) are necessary in vaccine growth, as a result of they will function strong predictors of future vaccine efficacy whether or not they’re derived from animal experiments or scientific trials and whether or not the tip factors contain safety from an infection or a discount in illness severity. There are nuances to the identification of CoPs in population-wide research that we can’t deal with right here (69, 70). The current macaque problem research usually are not sufficiently powered and usually are not wide-ranging sufficient in scope to permit the identification of CoPs with excessive confidence. The few makes an attempt to establish CoPs have pointed towards a predominant position for NAbs, which isn’t surprising, however it’s untimely to conclude that NAb titers on the time of problem (i.e., in people, virus publicity) inform the whole story. The doable position of recall responses (i.e., T and B cell reminiscence) in clearing a transient an infection has solely been addressed within the INOVIO DNA vaccine examine, which concerned the longest delay between immunization and problem (Desk 3). Even in that experiment, the quantity of knowledge obtainable is kind of restricted (7).

Regardless of the restrictions of the obtainable information, we sought hints of CoPs. Thus, we analyzed the relationships between, on the one hand, binding antibody and NAb titers on the prechallenge peak or inside 2 weeks earlier than problem and, then again, VL reductions in vaccinated animals in contrast with controls (summed for 2 locales of sampling and primarily based solely on subgenomic RNA; Tables 2 and three). We discovered no tangible nonparametric (Spearman) optimistic correlations of any significance between VL discount and any antibody parameters, which isn’t surprising given the variety of variables between the totally different experiments. We had been additionally unable to establish any constant relationship between the problem virus dose or supply route and the diploma of safety. The problem dose was not a constant predictor of the magnitude or period of excessive VLs within the management animals, however we must always keep in mind that the problem virus shares signify one other variable, as does the subspecies (and sources) of the animals concerned. Nonetheless, the extensive variation in problem dose between experiments shouldn’t be ignored. Would a vaccine that protected in opposition to a comparatively low problem dose be as protecting in opposition to the 100-fold larger dose utilized in different experiments? Or would its protecting capabilities degrade underneath these situations? Experimental situations yielding excessive VLs in management animals might impede full safety whereas giving the potential for higher VL reductions. The smallest VL lower within the vaccine group in comparison with management was seen within the INOVIO DNA vaccine experiment that concerned one of many lowest problem doses (Desk 3) (7). Within the report on the Ad26.COV2 vaccine, there have been indications of sterilizing immunity in opposition to an intermediate problem dose (4).

In 4 research, some teams of vaccine recipients appear to be utterly protected, or practically so (4, 6, 10, 11). In a single experiment, no anamnestic antibody or mobile immune responses had been detected within the protected animals, which urged that immunity was sterilizing (4). Nevertheless, in one other examine, there have been anamnestic immune responses in animals with undetectable VLs, which is extra indicative of incomplete however aborted an infection (6). Anamnestic responses weren’t analyzed within the different reviews (10). In two different instances, lung lobes within the vaccine teams the place safety was strongest had been freed from viral RNA 7 days after an infection, which contrasted with the excessive ranges discovered within the decrease lobes of management animals (1, 2). The standards for sterilizing immunity, or no less than full safety in opposition to persistent an infection, are neither outlined nor standardized within the SARS-CoV-2 animal mannequin subject, which additionally extends into different safety problem techniques involving small animals and each vaccines and antiviral antibodies (34, 51, 64–66).

Inside particular person research there are fewer confounding components than in a cross-study meta-analysis. Some proof was offered that antibodies had been the CoP within the Moderna mRNA vaccine examine (8). Within the report on the Janssen Ad26.COV2 vector vaccine, evaluating numerous antibody and T cell responses with an infection outcomes (as judged by VLs) recognized NAbs because the strongest CoP, with some doable contribution from Ab effector capabilities reminiscent of antibody-dependent mobile phagocytosis and antibody-dependent activation of pure killer cells. In distinction, T cell responses [measured by ELISpot or intracellular cytokine staining (ICS)] didn’t correlate with safety (4). Comparable inferences about an antibody however not a T-cell–related CoP emerged from the experiments involving DNA-plasmid immunizations (6). Nevertheless, even when important variations had been recognized, the ranges of the assorted measurements had been typically overlapping between utterly and partially protected animals (4, 6). This diploma of variation compromises makes an attempt to establish the edge response required for defense, notably within the examine of the Ad26 vector vaccine (4). General, the obtainable proof from macaque problem experiments does level towards a protecting position for antibody-based immunity (in all probability NAbs), however to not the extent {that a} protecting titer might be inferred after which extrapolated to the end result of human efficacy trials.

“s protein vaccine”

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