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Early post-infection treatment of SARS-CoV-2 infected macaques with human convalescent plasma with high neutralizing activity had no antiviral effects but moderately reduced lung inflammation [1]
['Koen K. A. Van Rompay', 'California National Primate Research Center', 'University Of California', 'Davis', 'California', 'United States Of America', 'Department Of Pathology', 'Microbiology', 'Immunology', 'Katherine J. Olstad']
Date: 2022-07
Early in the SARS-CoV-2 pandemic, there was a high level of optimism based on observational studies and small controlled trials that treating hospitalized patients with convalescent plasma from COVID-19 survivors (CCP) would be an important immunotherapy. However, as more data from controlled trials became available, the results became disappointing, with at best moderate evidence of efficacy when CCP with high titers of neutralizing antibodies was used early in infection. To better understand the potential therapeutic efficacy of CCP, and to further validate SARS-CoV-2 infection of macaques as a reliable animal model for testing such strategies, we inoculated 12 adult rhesus macaques with SARS-CoV-2 by intratracheal and intranasal routes. One day later, 8 animals were infused with pooled human CCP with a high titer of neutralizing antibodies (RVPN NT 50 value of 3,003), while 4 control animals received normal human plasma. Animals were monitored for 7 days. Animals treated with CCP had detectable but low levels of antiviral antibodies after infusion. In comparison to the control animals, CCP-treated animals had similar levels of viral RNA in upper and lower respiratory tract secretions, similar detection of viral RNA in lung tissues by in situ hybridization, but lower amounts of infectious virus in the lungs. CCP-treated animals had a moderate, but statistically significant reduction in interstitial pneumonia, as measured by comprehensive lung histology. Thus overall, therapeutic benefits of CCP were marginal and inferior to results obtained earlier with monoclonal antibodies in this animal model. By highlighting strengths and weaknesses, data of this study can help to further optimize nonhuman primate models to provide proof-of-concept of intervention strategies, and guide the future use of convalescent plasma against SARS-CoV-2 and potentially other newly emerging respiratory viruses.
The results of treating SARS-CoV-2 infected hospitalized patients with COVID-19 convalescent plasma (CCP), collected from survivors of natural infection, have been disappointing. The available data from various studies indicate at best moderate clinical benefits only when CCP with high titer of neutralizing antibodies was infused early in infection. The macaque model of SARS-CoV-2 infection can be useful to gain further insights in the value of CCP therapy. In this study, animals were infected with SARS-CoV-2 and the next day were infused with pooled human convalescent plasma, selected to have a very high titer of neutralizing antibodies. While administration of CCP had minimal effects on reducing virus replication in the respiratory tract, it significantly reduced lung inflammation. These data, combined with the results of monoclonal antibody studies, emphasize the need to use products with high titers of neutralizing antibodies, and guide the future development of antibody-based therapies.
Funding: This study was funded by California National Primate Research Center: Office of Research Infrastructure Program, Office of The Director, National Institutes of Health under Award Number P51OD011107. SI received funding from the National Institute of Allergy and Infectious Diseases, grants 1R21AI143454-02S1 and 3RF1AG061001. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
To further explore the potential benefit of CCP, and also to further validate the nonhuman primate model of SARS-CoV-2 to explore such passive immunotherapeutic interventions, the current study tested a pooled very high titer human CCP administered to rhesus macaques one day after high-dose virus inoculation, and compared it to animals treated with pooled control plasma. While administration of CCP had minimal effects on reducing virus replication in the respiratory tract, it had a statistically significant effect on reducing lung inflammation. Overall, however, CCP therapy was inferior compared to a monoclonal antibody-based strategy previously tested in this animal model under the same experimental conditions.
Relevant animal models can be very helpful in understanding the efficacy of CCP and guiding this decision process. SARS-CoV-2 infection of nonhuman primates is a relevant animal model, because, despite generally no or mild overt clinical signs, it recapitulates many of the key features of human infection including high levels of virus replication, immunological responses to infection, and the development of interstitial pneumonia [ 24 – 30 ]. The macaque model has been used to demonstrate the clear therapeutic efficacy of monoclonal antibodies [ 31 – 33 ]. Therapeutic studies with CCP in nonhuman primates have given mixed results. A pooled human CCP with moderate antibody titer given to rhesus macaques one day after virus inoculation failed to reduce virus replication [ 24 , 34 ]). In contrast, administration of a high-titer CCP, derived from convalescent African green monkeys, to African green monkeys 10 hours after inoculation had some therapeutic benefits, although variability and small group sizes limited statistical significance [ 35 ].
Considering the increased development and availability of potent neutralizing monoclonal antibodies, which despite high manufacturing costs, can be administered at high doses and have clearly proven efficacy [ 22 , 23 ], it is unclear whether further investment in high-titer CCP-based strategies is scientifically and logistically merited, or what directions should be explored to make CCP more efficacious or cost-effective. For example, there is consideration of the potential collection and use of CCP derived from previously infected donors who were later vaccinated, which results in increased titers and breadth or neutralizing antibody reactivity against variants of concern (transfusion of CCP from vaccinated donors, including from vaccine-boosted previously infected donors, is not currently allowed according to the FDA EUA). Regardless, lessons gained from experience with SARS-CoV-2 CCP can be beneficial for rapid responses in future pandemics with other respiratory infectious agents.
Major hurdles for high-titer CCP-based strategies are that (i) most convalescent individuals don’t develop high titers of neutralizing antibodies [ 18 , 19 ], (ii) the window of opportunity for CCP collection is limited, as neutralizing antibody titers decline over time [ 20 , 21 ], and (iii) passive antibodies are unavoidably diluted in the recipient after CCP infusion. Thus, CCP-based therapies require careful screening of many plasma units to identify the relatively few donors who have a sufficiently high titer of antiviral antibodies. Currently the FDA defines high-titer CCP as having a neutralization titer of ≥250 (in the Broad Institute’s neutralizing antibody assay) or corresponding S/C cutoff thresholds defined by FDA for high-throughput binding antibody assays (e.g., ≥ 23 on the Ortho VITROS spike IgG assay, which was the first assay to be qualified for release of CCP by FDA) [ 15 ].
The early Emergency Use Authorization (EUA) by the Food and Drug Administration (FDA) of CCP for SARS-CoV-2 therapy in August 2020 was based on early promising results suggesting that the known and potential benefits of CCP outweighed any known and potential risks [ 5 ]. However, most of these early studies were observational, hindered by biases and other limitations. Since then, randomized clinical trials (including a meta-analysis) have indicated no therapeutic benefits of the average CCP, or at best, minimal benefits for CCP with high antiviral antibody titers given during the early stages of disease, prior to seroconversion including development of endogenous neutralizing antibodies [ 6 – 14 ]. Because of these later results, the FDA revised the EUA for CCP in February 2021, limiting the use to only high-titer CCP, and only in hospitalized patients who are early in the disease course or in those with impaired humoral immunity who cannot produce an adequate antibody response to control SARS-CoV-2 replication [ 15 , 16 ]. Recent WHO guidelines recommend against the use of CCP except in the context of randomized clinical trials with severely ill patients [ 17 ].
In early 2020, the rapid surge in infection and hospitalization rates led to an urgent need for therapeutic interventions. This urgency sparked a high interest in collection and infusion of COVID-19 convalescent plasma (CCP), collected from people who had recovered from infection and had made antiviral antibodies, with particular focus on infusing CCP into recently infected patients with the hope of ameliorating their disease course. There were multiple rationales for the use of CCP. Despite variable evidence of efficacy, CCP therapy is a classic immunotherapy that has been applied to the prevention and treatment of many infectious diseases for more than a century, including more recently SARS, MERS, and the 2009 H1N1 influenza pandemic (reviewed in [ 2 – 4 ]). In addition, during a pandemic where many infected people survive and are willing to donate plasma, CCP can be collected by blood collection organizations (BCOs) and made readily available at relatively low cost. Finally, the reactivity of CCP evolves with the pandemic, as antibodies derived from recent convalescent survivors are expected to recognize recently circulating variants.
(A). Spearman r correlation matrix in heatmap format. For this analysis, lung pathology scores are the interstitial cellularity scores (from Fig 7 ). Lung ISH are the in situ hybridization data of Fig 6 . Peak NT 50 represents the peak neutralizing antibody titers up to day 5 (i.e., prior to possible de novo antibody responses). VITROS anti-spike total Ig represents the peak value for each animal (i.e., day 2; Fig 2 ). Nasal, oropharyngeal and BAL sgRNA values are based on AUC of the data in S7 Fig . Clinical scores (sedated and cage-side) are the tabulated scores of each animal over the 7-day observation period ( Fig 3C and 3F ). (B) Correlation between neutralizing antibody peak NT 50 values and lung pathology scores (Spearman r = -0.87; p < 0.0005). The labels next to each symbol indicate the individual animal.
A multivariable analysis with correlation matrix was performed on the main data sets, including neutralization activity, virus replication, lung pathology scores and clinical scores. With the caveat of the small group sizes, lung pathology scores had a highly significant inverse correlation with peak serum NT 50 values (Spearman r = -0.87; p < 0.001), followed by peak plasma VITROS anti-spike total Ig levels (Spearman r = -0.59, p = 0.046). In contrast, sgRNA viral loads in upper and lower respiratory tract secretions and lung in situ hybridization scores correlated poorly with lung pathology scores ( Fig 8 ). When only the data of the CCP-treated animals were analyzed, the negative correlation between NT 50 peak titers and lung pathology scores was slightly reduced (Spearman r = -0.71, p = 0.059), while NT 50 peak titers correlated negatively with cage-side clinical scores (Spearman r = -0.77, p = 0.04); S9 Fig .
A . Interstitial cellularity was evaluated on 7 lung lobes and an average score was tabulated as outlined in the Materials and methods section. Lines indicated mean values. The CCP group had significantly lower scores than the control group (p = 0.006; unpaired t-test). B-F . Interstitial cellularity score assigned to random x40 fields is based on the number of cells expanding the alveolar interstitium. Representative x40 images are shown. B . Grade 0: normal lung with thin acellular alveolar septae (animal CCP-7). C . Grade 1: alveolar interstitium expanded by 1 to 2 cells (animal CCP-1). D . Grade 2: alveolar interstitium expanded by 2 to 4 cells (animal CCP-1) E . Grade 3: alveolar interstitium expanded by 4 to 6 cells. (animal Co-3) F . Grade 4: alveolar interstitium expanded by more than 6 cells (animal Co-3).
To evaluate infection-induced lung pathology, a comprehensive histology scoring system, described in detail in the Materials and methods and validated in an earlier study [ 33 ], was used to tabulate interstitial cellularity scores. This scoring system takes into consideration (i) interstitial pneumonia as the most striking and consistent lesion in the lungs of SARS-CoV-2 infected macaques at 7 days of infection, (ii) the multifocal to locally extensive highly random distribution of the lesions and absence of distinct borders, (iii) the requirement of x40 magnification for accurate evaluation of the severity of the lesions. An average of 1208 microscopic fields (range: 710–1634) per animal, representing all 7 lung lobes, were graded from 0 to 4 in a blind analysis to tabulate an overall interstitial cellularity score.
In situ hybridization (RNAScope) was used to detect viral RNA in lung sections. (A) For each animal, the number of positive cells was counted in approximately 20 fields of right caudal lung lobe. Lines indicate median values. There were no differences between both study groups (p = 0.89, Mann Whitney test). (B) Example of lung section of animal CCP-2, with the arrows indicating RNA-positive cells.
In situ hybridization (using RNAScope) was performed on fixed lung specimens of the right caudal lung lobe; the number of viral RNA-positive cells was counted and expressed per surface area. There was no difference between the 2 experimental groups ( Fig 6A ). Viral RNA did not necessarily co-localize with lesions and could be detected in areas of the pulmonary parenchyma that were not inflamed ( Fig 6B ).
(A) Infectious virus titers in BAL supernatant (expressed as plaque-forming units (PFU) per ml) did not show any difference between the 2 study groups for any of the time points of BAL collection. Lines indicate median values. The dotted line indicates the limit of detection (LOD). (B) Infectious virus titers in lung tissues. For each animal, two snapfrozen lung specimens (one sample per left and right caudal lung lobe) were tested for infectious virus. The only 3 samples with detectable virus were all right caudal lobe samples of 3 control animals, with detection of 1 plaque; taking the dilution factor of the assay and weight of the original tissue specimen into account, this was then expressed as PFU per mg lung tissue. Comparison of both groups revealed a significant difference (p = 0.028, Mann-Whitney test).
Infectious virus titers were measured by plaque assay on BAL supernatants and on frozen lung specimens collected at time of euthanasia. There was no difference in infectious titers in BAL samples ( Fig 5A ). Infectious virus was detected in the right caudal lobe of 3 of the 4 control animals, but not in any lung specimens of the CCP-treated animals ( Fig 5B ; p = 0.028, Mann-Whitney test).
(A) to (C) Time course of median viral subgenomic (sg) RNA copies (with error bars showing the range) in nasal swabs, oropharyngeal swabs and BAL samples, respectively. Red and black arrows indicate time of virus inoculation and infusion of plasma (control plasma or CCP) on days 0 and 1, respectively. sgRNA was measured by RT-qPCR and expressed relative to cellular mRNA of the housekeeping gene PPIA (as indicator of the cellular content in the sample tested) by plotting the difference in CT values; thus, a larger difference indicates less virus replication. The dotted line indicates the limit of detection (LOD). More details are provided in S7 and S8 Figs.
sgRNA levels are considered the best evidence of active virus replication. Analysis of sgRNA in nasal and oropharyngeal swabs and BAL demonstrated that treatment with CCP at 1 day after infection did not have any detectable effect on virus replication in the upper or lower respiratory tracts, ( Fig 4 , S7 and S8 Figs). The data on total vRNA and gRNA levels gave similar conclusions ( S7 Fig ).
Nasal swabs, oropharyngeal swabs, and broncho-alveolar lavages (BAL) were collected regularly for viral load analysis. Samples were tested by RT-qPCR for total viral RNA (vRNA, N target), genomic viral RNA (gRNA, ORF1a target), subgenomic viral RNA (sgRNA, leader/N target), and cellular mRNA of the housekeeping gene PPIA (Peptidylprolyl Isomerase A). In general, and as previously shown [ 24 , 33 ], the relative ratios of the 3 types of viral RNA’s were quite consistent in the samples (vRNA > gRNA > sgRNA).
Analysis of immune subsets in whole blood revealed rapid dynamic changes in frequencies of innate immune cells as previously reported [ 24 ]. Relative frequencies of innate immune subsets in blood—neutrophils, proinflammatory monocytes, myeloid dendritic cells (mDC), plasmacytoid dendritic cells (pDC)—changed rapidly following infection in both experimental groups ( S6 Fig ). Assessment of T cell responses demonstrated a net decrease in naive CD4+ T cell frequencies at day 1 and day 7 post infection, while CD8+ T cell frequencies remained unchanged. Following infection, frequencies of both central memory (CD95+ CD28+) CD4+ and CD8+ T cells increased while effector memory (CD95+ CD28-) frequencies declined indicative of antigen driven activation and migration of T cells. In support of this, frequencies of Ki-67+ PD-1+ CD4 T cells were significantly increased at day 7 in both groups. Altogether, the data are consistent with infection-induced activation of the innate and adaptive arms in both the convalescent and normal plasma groups.
Following infection, most animals had a transient increase in C-reactive protein, ALT and AST, peaking on day 2, and a transient increase of several cytokines and chemokines, including IL-6, MCP-1, Eotaxin, I-TAC, IL-1RA and IP-10, generally peaking on day 1 or 2 ( S3 and S4 Figs). Other cytokines and chemokines did not show consistent changes ( S5 Fig ). Overall, there were no discernible differences in these parameters between the 2 animal groups.
Similarly, due to the mild disease course, there were no differences between the control and CCP-treated animals for clinical scoring performed at time of sedation ( Fig 3D–3F ; p = 0.41, Mann Whitney; S2 Fig ). All animals had stable weights throughout the observation period, consistent with an adequate appetite. Three animals (2 CCP-treated animals and 1 control animal) had at least one recording of mildly elevated rectal temperature (102.5–103 °F). None of the animals developed low oxygen saturation levels (Sp02 < 95%).
Red and black arrows indicate time of virus inoculation and plasma administration on days 0 and 1, respectively. (A, B, D, E) Daily clinical scores based on cage-side observations and sedated measurements for each animal of the 2 study groups; the maximum daily score possible is 22 (for cage-side observations) and 27 (for sedated observations). (C, F) For each animal, the total of clinical scores over the 7-day period was tabulated. Comparison of the 2 groups revealed no detectable therapeutic benefits of the CCP treatment (p ≥ 0.28, Mann-Whitney).
Animals were scored daily for several clinical parameters by cage-side observations. Overall, clinical signs were absent or mild-to-moderate (daily scores ≤ 4 out of a maximum of 22) and consisted mostly of nasal discharge. The highest daily score of 4 was recorded for CCP-treated animal CCP-8, which had a few observations of slightly increased abdominal breathing. When the sums of the daily scores from day 0 to 7 were tabulated for each animal, no significant difference was observed between the 2 treatment groups ( Fig 3A–3C ; p = 0.28 Mann-Whitney).
Animals were inoculated on day 0 (red arrow) and administered either COVID convalescent plasma (CCP) or control (Co) plasma on day 1 (black arrow). (A) Neutralizing activity was measured in serum samples of the animals using a RVPN assay, with estimation of the titer to get 50% inhibition (NT 50 ). For comparison, the NT 50 titer of the administered CCP was 3,003. Samples with undetectable titers are presented at the limit of detection (1:40). (B) VITROS anti-spike total Ig is expressed as the ratio of signal over cut-off (S/CO). A value of ≥1 indicates reactivity. The S/CO value of the administered pooled CCP was 684.
One day after infusion of plasma, all CCP-treated animals had detectable neutralizing activity in serum samples, as determined by RVNP and binding antibody assays ( Fig 2 and S2 Table ). Between day 2 to 5, peak 50% neutralization titers (NT 50 ) had a median value of 155, while NT 80 values were near or below the assay’s limit of detection (titer of 40; S2 Table ). By day 7, some animals, including one control animal, had an increase in neutralizing activity in serum, indicative of a de novo antibody response. Similarly, starting one day after the infusion of CCP, all CCP-treated animals had detectable anti-spike immunoglobulins (as measured by the VITROS spike Total Ig assay), and reactivity to many SARS-CoV-2 antigens (as detected by the coronavirus antigen micro-array assay), which persisted throughout the observation period ( S2 Table and Fig 2B and S1 Fig ). Overall, the magnitude of the signals was as expected, based on the CCP being diluted ~1:60-fold upon transfusion in the animals.
Twelve young adult macaques were inoculated with a high dose (2.5 x10 6 PFU) of a Washington isolate of SARS-CoV-2 by the intratracheal and intranasal routes. One day later, eight animals received an intravenous infusion with the pooled CCP (4.8 ml/kg), while the other four animals were treated with control plasma (4.8 ml/kg). This dose is in the same range as administration of one unit of plasma (200–250 ml) to a human adult. Animals were monitored closely by clinical observation, radiographs, and regular sample collection, and were euthanized for tissue collection on day 7 ( Fig 1C ).
The pooled CCP and control plasma were also tested on a coronavirus antigen micro-array assay that detects antibodies against antigens of SARS-CoV-2, SARS-CoV-1, MERS and seasonal coronaviruses. As expected, the pooled CCP (but not the control plasma) had very high level reactivity against most SARS-CoV-2 antigens, while both CCP and normal plasma pools had similar low cross-reactivity to other coronaviruses ( Fig 1B ).
Two pools of human plasma, COVID convalescent plasma (CCP) and normal plasma, were prepared by mixing plasma of convalescent patients or pre-pandemic uninfected donors, respectively. (A) The 2 plasma pools were characterized for neutralizing antibody titers (NT50 and N80 values), for total spike Ig (by VITROS assay) and IgG (by VITROS Anti-SARS-CoV-2 IgG Quantitative Test; units are BAU/ml). (B) The two plasma pools were also tested by coronavirus microarray assay (COVAM), and signal values are graphed as a heatmap. While the CCP had high reactivity to most SARS-CoV-2 antigens, cross-reactivity of the normal plasma pool to SARS-CoV-2 antigens was very low. Both plasma pools had similar reactivity to non-SARS-CoV-2 antigens. (C) Twelve adult rhesus macaques were inoculated on day 0 with SARS-CoV-2 by both intratracheal and intranasal routes. On day 1, eight animals received a single intravenous infusion with pooled CCP, while the other 4 animals received pooled normal control plasma. Animals were monitored closely for clinical signs (both cage-side and sedated observations) with regular collection of radiographs and samples to monitor infection and disease. On day 7, animals were euthanized for detailed tissue collection and analysis.
High-titer human CCP was prepared by mixing 3 plasma units (from 3 different donors), identified to have the highest titers of neutralizing antibodies (as determined by the reporter viral particle neutralization (RVPN) assay), and the highest reactivity of anti-spike total Ig (as determined by the Ortho VITROS IgG assay) [ 21 ]. The pooled CCP had high antiviral activity as demonstrated by NT 50 , NT 80 and VITROS S/CO values of 3,003, 1,113 and 684, respectively ( Fig 1A and S1 Table ). Based on testing a dilution in the VITROS Anti-SARS-CoV-2 IgG Quantitative Test (which measures antibodies against S1 protein and is calibrated against NIBSC/WHO standards), the CCP pool was found to have a high titer of 775 binding antibody units (BAU)/ml, well above the 200 BUA/ml upper limit of the measuring interval of NIBSC/WHO standards.
Discussion
The current study provides insights on the efficacy and limitations of CCP therapy against SARS-CoV-2 replication and COVID-19 disease, as well as the opportunities and challenges associated with the use of a nonhuman primate model in testing passive immunotherapy strategies.
To maximize the use of the nonhuman primate model of SARS-Cov-2 to test therapeutic strategies, animals are generally inoculated with a high dose of virus. A limitation of this nonhuman primate model of SARS-CoV-2 is that most animals display no or mild clinical signs, and have minor radiographic changes, which limits the use of these markers to evaluate intervention strategies. Accordingly, in this animal model, the primary and most sensitive read-outs for the effectiveness of therapeutic strategies consist of measuring their impact on virus replication and lung inflammation, as both kinds of markers are generally readily detectable after high-dose virus inoculation even in the absence of any overt clinical disease. Despite considerable individual variability in these markers, this animal model has been validated to detect benefits of potent therapeutic strategies, such as monoclonal antibodies [33].
In the current study, we demonstrate that administration of a pooled human CCP with high titer of neutralizing and spike-binding antibodies, administered one day after virus inoculation, conferred therapeutic benefits to SARS-CoV-2 infected macaques in terms of moderately reduced interstitial pneumonia, despite limited effects on markers of virus replication. Therapeutic effects of CCP with high titers of neutralizing antibodies were recently also described in Syrian hamsters [36].
In this study, high-titer CCP had no detectable effect on reducing viral RNA and infectious virus levels in mucosal samples, or on the number of viral RNA-positive cells in lung sections. The only difference was in infectious virus in lung samples, because unlike the control animals (where 3 of the 4 animals had one lung lobe with detectable infectious virus), none of the CCP-treated animals had any detectable infectious virus in lung samples. However, one cannot rule out that in the CCP-treated animals, a low concentration of antiviral antibodies present in the lung specimen may have interfered with viral replication in the in vitro plaque assay, particularly as infectious virus levels were already near the limit of detection in the control animals on day 7. Thus, overall, the effects of CCP on reducing markers of viral replication were, at best, minimal.
The minimal effects of the CCP on markers of virus replication is likely multifactorial, with insufficient antiviral activity as the primary explanation, but influenced by additional experimental factors. Although we used a CCP with high neutralizing activity in vitro, the antibodies became diluted so much upon transfusion that by the time they reached mucosal sites, their concentration was probably too low to have a drastic impact on reducing virus replication in vivo. In this context, it is important to note that we inoculated animals with a very high dose of SARS-CoV-2, to induce rapid wide-spread infection of upper and lower respiratory tract, with peak virus replication occurring within the first 1–2 days. Having high levels of viral replication at the time of CCP administration sets a high bar to detect efficacy, especially as it takes time for passively infused antibodies to distribute and reach peak concentrations at mucosal sites. The difficulty to detect a difference was likely exacerbated by the considerable variability in virus levels in mucosal samples and tissue specimens of SARS-CoV-2 infected animals, including untreated control animals, as observed in many other studies [26,27,31,37]. This high variability is likely a combination of individual variability in virus replication, but also the variability inherently associated with sampling mucosal secretions or tissues, which represents a snapshot in time of viral shedding or replication at a limited mucosal surface. Thus, while small animal group sizes can still allow detection of large differences in virus replication caused by very potent antiviral strategies including passive immunotherapy with monoclonal antibodies [33], they lack the power to detect relatively mild-to-moderate antiviral effects.
In contrast to the minimal effects on markers of virus replication, CCP treatment had a modest but statistically significant beneficial effect on reducing lung inflammation. The reason that the relatively modest difference (~17%) in interstitial cellularity scores between the study groups was statistically significant, despite the relatively small group sizes, can be attributed to the very comprehensive and extensive scoring system, in which the evaluation of numerous microscopic fields per animal provides a relatively precise assessment of the overall extent of interstitial pneumonia at 7 days of infection. Despite its advantages of being highly rigorous and robust, this scoring system has the drawback of being labor-intensive, which precludes application on large-scale studies. Thus, future efforts can focus on further refining it, by simplification (i.e., pathologist-driven scoring of fewer fields or fewer lobes but achieving statistically similar reliable results) and/or automation (i.e., computer-generated scores).
There is precedence for a relative dissociation between SARS-CoV-2 virus replication, particularly in the upper respiratory tract, and pulmonary lesions, as demonstrated in several therapeutic studies in SARS-CoV-2 infected macaques [27,38]. Dose-range vaccine studies in macaques found that higher antibody levels were needed to reduce virus replication in the upper airways than in the lower respiratory tract [39]; this can explain recent observations that some vaccinated people with breakthrough infections with the SARS-CoV-2 delta variant can have similar viral loads in upper respiratory tract as unvaccinated people, but yet, remain at much reduced risk for severe illness and hospitalization [40,41]. Finally, as confirmed in the current study, it has been demonstrated in the lungs of SARS-COV-2 infected macaques that the virus does not necessarily co-localize with the lesions and can be found in areas of the pulmonary parenchyma that are not inflamed [29]. Altogether these observations underscore the importance of pulmonary histopathology as a key endpoint when evaluating the efficacy of therapeutics.
The data of the current study help to further define neutralizing activity as a correlate of efficacy for antibody-based antiviral therapeutic strategies against SARS-CoV-2. A previous study used the same experimental design as the current study, except that one day after virus, instead of CCP, animals received a combination of 2 potent monoclonal antibodies [33]. In that study, neutralizing antibody titers in serum after infusion were ~ 2 to 3 logs higher than those observed in the current study. Despite similarly small group sizes, the antibody-treated animals had statistically significant reductions in virus replication, clinical signs, and interstitial pneumonia (~ 50% reduction in interstitial cellularity scores) compared to control antibody-treated animals. Comparison of these 2 studies, with the lung histology scoring performed by the same pathologists, revealed that animals treated with monoclonal antibodies had significantly lower lung pathology scores than the CCP-treated animals in the current study (S10 Fig), indicating the superiority of monoclonal antibodies above CCP to treat early SARS-CoV-2 infection.
In the current CCP study, peak neutralizing antibody NT 50 values in serum the first few days after infusion were ~150, which, considering the marginal efficacy observed in the histology scoring, helps to set a threshold for neutralizing antibody titers in the recipient to have a chance at any therapeutic benefits. Although direct comparison of neutralization data across studies is difficult due to differences in assays and other laboratory conditions, the threshold value observed in this current study is consistent with recent findings from other animal and human studies. A study that used purified IgG derived from convalescent macaques found that a NT 50 titer between 50 and 500 in the recipient animals was the threshold to see an effect on reducing virus replication, although no lung histology scoring was reported [42]. In a study with African green monkeys, the administration of a high-titer CCP, derived from convalescent African green monkeys, administered 10 hours after a moderate-dose virus inoculation, resulted in live-virus plaque reduction neutralization titers 50% (PRNT 50 ) of ~ 128 in the recipient animals, that were associated with reduced virus replication and histology, although differences were statistically not significant, likely due to variability and small group sizes [35]. In human studies, it has been difficult to set a threshold for neutralizing activity after CCP infusion, as generally infusions were performed later in the course of infection (i.e. when de novo antibody responses were already being generated), or studies did not report neutralization titers in the CCP recipients [8,10].
The combined results of these studies provide further guidance to what future, if any, CCP can have in the clinic for early treatment of SARS-CoV-2 infected people. First of all, as very few people who recover from COVID-19 develop persistently high neutralizing antibody titers, the use of CCP from such donors faces increasing logistical and scientific hurdles, especially considering the increased availability of potent monoclonal antibodies which can be administered at high doses in relatively small volumes. However, recent studies have demonstrated that COVID-19 survivors who subsequently received a SARS-CoV-2 vaccine made very strong booster immune responses, which also neutralize currently circulating variants of concern [43–49]. Thus, vaccinated COVID-19 recovered subjects are likely to be a much better source of CCP. In addition, although expensive to manufacture and thus less feasible in resource-poor areas, purified IgG derived from CCP could also allow higher antibody delivery in a smaller volume, and thus lead to better efficacy.
The current study also helps to further validate and strengthen the nonhuman primate model of SARS-CoV-2 infection and COVID-19. Although the typical disease course of SARS-CoV-2 infection in young, otherwise healthy macaques is mild, and despite a limited number of animals, a detailed analysis was able to detect relatively mild-to-moderate therapeutic benefits of high-titer CCP administration. These findings will be relevant for future pandemics with newly emerging respiratory viruses, as the rapid development of relevant nonhuman primate models with proper monitoring and scoring systems can speed up testing the safety and efficacy of antiviral strategies including CCP and monoclonal antibodies to generate the data that can guide the design of clinical trials.
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