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The objective of this study was to compare, by qPCR, the circulating blood parasite load of Trypanosoma cruzi in the buffy coat, and in whole blood mixed with boiled and unboiled guanidine hydrochloride-EDTA buffer, of individuals with chronic ChD. The concentration and purity of DNA were evaluated in a Nanodrop Denovix DS-11FX Series Spectrophotometer (DeNovix Inc., Wilmington, NC, USA). The parasite load was determined with the Taqman® qPCR system using a Stratagene Mx3000P thermocycler (Agilent Technologies, Santa Clara, CA, USA) with Cruzi 1 and Cruzi 2 satellite primers. Student’s t-test with Bonferroni correction, Chi-squared (χ2) tests and Spearman’s correlation coefficient were applied. The concentration and purity of DNA were higher in the buffy coat. Parasite DNA was detected and quantifiable in the three types of samples in seven patients, without statistically significant differences in the parasite load obtained. Higher correlations were found between the total DNA concentrations and the parasite loads obtained in the samples of the buffy coat.

ABSTRACT: Objectives: This study aimed to describe the electrocardiographic and echocardiographic status of chronic Chagas disease (cChD) patients treated with nifurtimox. Methods: An observational study was performed in 146 cChD patients followed over a mean of 7.9 years. Results: Of the 146 patients, 41 (28.1%) with normal electrocardiogram (ECG) at baseline maintained this condition, 34 (23.3%) with altered ECG at baseline normalised the alterations, and 46 (31.5%) with ECG abnormalities at baseline maintained this condition [23 (15.8%) with small alterations]. Finally, 25 cases (17.1%) in indeterminate phase altered the ECG. Differences before and after follow-up (P < 0.001) were found. The percentage of beneficial treatment was different than expected by chance (Z = 4.8; P < 0.001) and the annual percentage of cases that developed ECG alterations was lower than that of a historical cohort of untreated patients (P < 0.001). An echocardiogram was performed in 68 patients with baseline ECG alterations. The ejection fraction (EF) was normal in 57 (83.8%) and abnormal in 11 (16.2%). In 38 patients with ECG abnormalities that did not progress after treatment, EF and segmental motility (SM) were normal in 31 (81.6%) and 26 (68.4%), respectively. In 17 patients with ECG abnormalities, EF and SM were normal in 15 (88.2%) and 14 (82.4%) cases, respectively. Conclusion: Less progression to cardiomyopathy compared with a historical untreated cohort as well as the EF/SM results in patients with abnormal ECG that did not progress and in indeterminate cChD that altered the ECG suggests a beneficial effect of nifurtimox.

Chagas disease (ChD) is a vector zoonosis native to the American continent caused by the protozoan parasite Trypanosoma cruzi; the biological vectors are multiple species of hematophagous insects of the family Triatominae. A relevant aspect in the host–parasite relationship is the identification of the various genotypes of T. cruzi called discrete typing units (DTU) that circulate in mammals and vectors. In Chile, it has been described that the DTUs TcI, TcII, TcV, and TcVI circulate in infected humans, vectors, and wild animals. Identifying DTUs has acquired clinical importance, since it has been suggested that different genotypes could cause distinct pathologies, circulate in different geographical areas, and present different sensitivities to trypanocidal drugs. In this study, circulating T. cruzi DTUs in peripheral blood and Triatoma infestans dejections used in xenodiagnosis (XD) were amplified by qPCR in 14 Chilean patients with chronic ChD from highly endemic areas. More positive samples were detected by XD compared to peripheral blood samples, and 64.28% of the cases were simple infections and 35.72% mixed, with a statistically significant difference in the frequency of TcV DTU. This study would suggest that T. infestans from Chile is more competent to amplify one DTU over others, probably due to a process of co-evolution.

Chagas disease, a vector-borne parasitosis caused by Trypanosoma cruzi, is endemic to Latin America and has spread to other countries due to immigration of infected persons. It is estimated that 160,000 people are infected in Chile, most of them in the chronic phase and without etiological treatment. The infection is confirmed by conventional serological methods while molecular methods have become in valuable tools to evaluate parasitemia in treated and non-treated chronic Chagas disease patients. The objective of this study was to determine, by conventional Polymerase Chain Reaction, the presence of T. cruzi kinetoplastid DNA in peripheral blood samples from 114 adult individuals with confirmed chronic Chagas disease, before and 6.6 years (average) after treatment with nifurtimox. The samples were received and preserved in guanidine-EDTA until DNA purification. Conventional PCR assays were performed in triplicate with T. cruzi kinetoplastid DNA primers 121 and 122. The amplified products were fractionated by electrophoresis in 2% agarose gels. A 330 bp product represented a positive assay. 84.2% (96 cases) and 6.1% (7 cases) of the samples taken before and after the treatment, respectively, were positive. The McNemar test showed a statistically significant difference between the groups of samples (p

We evaluate the elimination of the microscopic stage of conventional xenodiagnosis (XD) to optimize the parasitological diagnosis of Trypanosoma cruzi in chronic Chagas disease. To this purpose we applied under informed consent two XD cages to 150 Chilean chronic chagasic patients. The fecal samples (FS) of the triatomines at 30, 60 and 90 days post feeding were divided into two parts: in one a microscopic search for mobile trypomastigote and/or epimastigote forms was performed. In the other part, DNA extraction-purification for PCR directed to the conserved region of kDNA minicircles of trypanosomes (PCR-XD), without previous microscopic observation was done. An XD was considered positive when at least one mobile T. cruzi parasite in any one of three periods of incubation was observed, whereas PCR-XD was considered positive when the 330 bp band specific for T. cruzi was detected. 25 of 26 cases with positive conventional XD were PCR-XD positive (concordance 96.2%), whereas 85 of 124 cases with negative conventional XD were positive by PCR-XD (68.5%). Human chromosome 12 detected by Real-time PCR used as exogenous internal control of PCR-XD reaction allowed to discounting of PCR inhibition and false negative in 40 cases with negative PCR-XD. Conclusion: PCR-XD performed without previous microscopic observation is a useful tool for detection of viable parasites with higher efficiency then conventional XD.

Werner AptUniversity of Chile, Faculty of Medicine, Santiago, ChileAbstract: Chagas disease must be treated in all its stages: acute, indeterminate, chronic, and initial and middle determinant chronic, due to the fact that DNA of the parasite can be demonstrated by PCR in chronic cases, where optical microscopy does not detect parasites. Nifurtimox (NF) and benznidazole (BNZ) are the drugs accepted to treat humans based upon ethical considerations and efficiency. However, both the drugs produce secondary effects in 30% of the cases, and the treatment must be given for at least 30–60 days. Other useful drugs are itraconazole and posaconazole. The latter may be the drug to treat Chagas disease in the future when all the investigations related to it are finished. At present, there is no criterion of cure for chronic cases since in the majority, the serology remains positive, although it may decrease. In acute cases, 70% cure with NF and 75% with BNZ is achieved. In congenital cases, 100% cure is obtained if the treatment is performed during the first year of life. In chronic acquired cases, 20% cure and 50% improvement of the electrocardiographic changes are obtained with itraconazole.Keywords: Chagas disease, treatment, nifurtimox, benznidazole, allopurinol, itraconazole, posaconazole

Congenital Chagas disease acquired special importance in Chile after the certification of the control of Triatoma infestans and transmission by blood donors affected with Trypanosoma cruzi. In order to establish adequate protocols for intervention and control in infected mother-neonate pairs in endemic zones of Chagas disease, we present partial results (2005-2008) of a pilot project which is being carried out in the Province of Choapa, IV Region, Chile, whose objectives are: determine the current prevalence of the disease in pregnant women, estimate the incidence of vertical transmission of T. cruzi to newborns, determine the lineages of the parasite present in mothers who do and do not transmit the disease, determine the prevalence of Chagas disease in maternal grandmothers of neonates and study placental histopathology. Preliminary results indicated that in this study period, 3.7% of the women who gave birth in the Province have Chagas disease and 2.5% of their newborns were infected. The most frequent T. cruzi genotypes found in mothers studied during pregnancy were TCI and TCIId, either alone or in mixed infections. A high percentage (74.3%) of the grandmothers studied was infected with the parasite. In 29 placentas from mothers with Chagas disease we observed edema, necrosis, fibrinoid deposits and slight lymphoplasmocyte infiltration. In three placentas we found erythroblastosis and in one of them amastigote forms of T. cruzi; this was one of the cases of congenital infection. The evaluation of the diagnostic and control protocols generated will allow us to determine if it has been possible to modify the natural history of vertical transmission of T. cruzi in Chile.

BACKGROUND: The Trypanosoma cruzi satellite DNA (satDNA) OligoC-TesT is a standardised PCR format for diagnosis of Chagas disease. The sensitivity of the test is lower for discrete typing unit (DTU) TcI than for TcII-VI and the test has not been evaluated in chronic Chagas disease patients. METHODOLOGY/PRINCIPAL FINDINGS: We developed a new prototype of the OligoC-TesT based on kinetoplast DNA (kDNA) detection. We evaluated the satDNA and kDNA OligoC-TesTs in a multi-cohort study with 187 chronic Chagas patients and 88 healthy endemic controls recruited in Argentina, Chile and Spain and 26 diseased non-endemic controls from D.R. Congo and Sudan. All specimens were tested in duplicate. The overall specificity in the controls was 99.1% (95% CI 95.2%-99.8%) for the satDNA OligoC-TesT and 97.4% (95% CI 92.6%-99.1%) for the kDNA OligoC-TesT. The overall sensitivity in the patients was 67.9% (95% CI 60.9%-74.2%) for the satDNA OligoC-TesT and 79.1% (95% CI 72.8%-84.4%) for the kDNA OligoC-Test. CONCLUSIONS/SIGNIFICANCE: Specificities of the two T. cruzi OligoC-TesT prototypes are high on non-endemic and endemic controls. Sensitivities are moderate but significantly (p = 0.0004) higher for the kDNA OligoC-TesT compared to the satDNA OligoC-TesT.

Chagas disease, produced by the protozoan Trypanosoma cruzi (T. cruzi), is one of the most frequent endemic diseases in Latin America. In spite the fact that in the past few years T. cruzi congenital transmission has become of epidemiological importance, studies about this mechanism of infection are scarce. In order to explore some morphological aspects of this infection in the placenta, we analyzed placentas from T. cruzi-infected mothers by immunohistochemical and histochemical methods. Infection in mothers, newborns, and placentas was confirmed by PCR and by immunofluorescence in the placenta. T. cruzi-infected placentas present destruction of the syncytiotrophoblast and villous stroma, selective disorganization of the basal lamina, and disorganization of collagen I in villous stroma. Our results suggest that the parasite induces reorganization of this tissue component and in this way may regulate both inflammatory and immune responses in the host. Changes in the ECM of placental tissues, together with the immunological status of mother and fetus, and parasite load may determine the probability of congenital transmission of T. cruzi.

From 1997 to 1999, we identified seven human cases of infection by fourth stage larvae of Pseudoterranova decipiens in Chile. All identified larvae were coughed up by the patients. Subjects were 10-55 years old; five were female. Some patients complained of coughing, expectoration, pharyngeal pain, nausea or anal and nasal pruritus. Larvae of three patients were coughed up from 36 h to 7 days after having eaten raw (cebiche or sushi) or lightly fried fish. P. decipiens has a marine life cycle. Infective third stage larva develop to adult stage in pinniped mammals. The nematode eggs are voided with the host faeces and develop and hatch releasing third stage larvae. Some crustaceans and fish act as hosts of third stage larvae. Man is an accidental host for third or fourth stage larvae.

BACKGROUND: PCR has evolved into one of the most promising tools for T. cruzi detection in the diagnosis and control of Chagas disease. However, general use of the technique is hampered by its complexity and the lack of standardization. METHODOLOGY: We here present the development and phase I evaluation of the T. cruzi OligoC-TesT, a simple and standardized dipstick format for detection of PCR amplified T. cruzi DNA. The specificity and sensitivity of the assay were evaluated on blood samples from 60 Chagas non-endemic and 48 endemic control persons and on biological samples from 33 patients, 7 reservoir animals, and 14 vectors collected in Chile. PRINCIPAL FINDINGS: The lower detection limits of the T. cruzi OligoC-TesT were 1 pg and 1 to 10 fg of DNA from T. cruzi lineage I and II, respectively. The test showed a specificity of 100% (95% confidence interval [CI]: 96.6%-100%) on the control samples and a sensitivity of 93.9% (95% CI: 80.4%-98.3%), 100% (95% CI: 64.6%-100%), and 100% (95% CI: 78.5%-100%) on the human, rodent, and vector samples, respectively. CONCLUSIONS: The T. cruzi OligoC-TesT showed high sensitivity and specificity on a diverse panel of biological samples. The new tool is an important step towards simplified and standardized molecular diagnosis of Chagas disease.

BACKGROUND Vector transmission of Trypanosoma cruzi appears to be interrupted in Chile; however, data show increasing incidence of Chagas' disease, raising concerns that there may be a reemerging problem. OBJECTIVE To estimate the actual risk in a changing world it is necessary to consider the historical vector distribution and correlate this distribution with the presence of cases and climate change. METHODS Potential distribution models of Triatoma infestans and Chagas disease were performed using Maxent, a machine-learning method. FINDINGS Climate change appears to play a major role in the reemergence of Chagas' disease and T. infestans in Chile. The distribution of both T. infestans and Chagas' disease correlated with maximum temperature, and the precipitation during the driest month. The overlap of Chagas' disease and T. infestans distribution areas was high. The distribution of T. infestans, under two global change scenarios, showed a minimal reduction tendency in suitable areas. MAIN CONCLUSION The impact of temperature and precipitation on the distribution of T. infestans, as shown by the models, indicates the need for aggressive control efforts; the current control measures, including T. infestans control campaigns, should be maintained with the same intensity as they have at present, avoiding sylvatic foci, intrusions, and recolonisation of human dwellings.

Multivalent antigen display is a fast-growing area of interest toward broadly protective vaccines. Current nanoparticle-based vaccine candidates demonstrate the ability to confer antibody-mediated immunity against divergent strains of notably mutable viruses. In coronaviruses, this work is predominantly aimed at targeting conserved epitopes of the receptor binding domain. However, targeting conserved non-RBD epitopes could limit the potential for antigenic escape. To explore new potential targets, we engineered protein nanoparticles displaying coronavirus prefusion-stabilized spike (CoV&#95;S-2P) trimers derived from MERS-CoV, SARS-CoV-1, SARS-CoV-2, hCoV-HKU1, and hCoV-OC43 and assessed their immunogenicity in female mice. Monotypic SARS-1 nanoparticles elicit cross-neutralizing antibodies against MERS-CoV and protect against MERS-CoV challenge. MERS and SARS nanoparticles elicit S1-focused antibodies, revealing a conserved site on the S N-terminal domain. Moreover, mosaic nanoparticles co-displaying distinct CoV&#95;S-2P trimers elicit antibody responses to distant cross-group antigens and protect male and female mice against MERS-CoV challenge. Our findings will inform further efforts toward the development of pan-coronavirus vaccines., (© 2023. Springer Nature Limited.)

SARS-CoV-2 Omicron is highly transmissible and has substantial resistance to neutralization following immunization with ancestral spike-matched vaccines. It is unclear whether boosting with Omicron-matched vaccines would enhance protection. Here, nonhuman primates that received mRNA-1273 at weeks 0 and 4 were boosted at week 41 with mRNA-1273 or mRNA-Omicron. Neutralizing titers against D614G were 4,760 and 270 reciprocal ID 50 at week 6 (peak) and week 41 (preboost), respectively, and 320 and 110 for Omicron. 2 weeks after the boost, titers against D614G and Omicron increased to 5,360 and 2,980 for mRNA-1273 boost and 2,670 and 1,930 for mRNA-Omicron, respectively. Similar increases against BA.2 were observed. Following either boost, 70%-80% of spike-specific B cells were cross-reactive against WA1 and Omicron. Equivalent control of virus replication in lower airways was observed following Omicron challenge 1 month after either boost. These data show that mRNA-1273 and mRNA-Omicron elicit comparable immunity and protection shortly after the boost., Competing Interests: Declaration of interests K.S.C. is an inventor on U.S. Patent no. 10,960,070 B2 and International Patent Application no. WO/2018/081318 entitled “Prefusion Coronavirus Spike Proteins and Their Use.” K.S.C. is an inventor on U.S. Patent Application no. 62/972,886 entitled “2019-nCoV Vaccine.” A.R.H., L.W., W.S., Y.Z., E.S.Y., J.R.M., P.D.K., M.R., N.J.S., and D.C.D. are inventors on U.S. Patent Application no. 63/147,419 entitled “Antibodies Targeting the Spike Protein of Coronaviruses.” L.P., A.V.R., B.N., D.V., A. Cook, A.D., K.S., H.A., and M.G.L. are employees of Bioqual. K.S.C., L.W., W.S., and Y.Z. are inventors on multiple U.S. Patent Applications entitled “Anti-Coronavirus Antibodies and Methods of Use.” G.-Y.C., G.S.-J., I.R., Y.-T.L., A.M., K.W., A. Carfi, and D.K.E. are employees of Moderna. M.S.S. serves on the scientific board of advisors for Moderna and Ocugen. The other authors declare no competing interests., (Published by Elsevier Inc.)

Knowledge about the impact of prior severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of the elderly on mRNA vaccination response is needed to appropriately address the demand for additional vaccinations in this vulnerable population. Here, we show that octogenarians, a high-risk population, mount a sustained SARS-CoV-2 spike-specific immunoglobulin G (IgG) antibody response for 15 months following infection. This response boosts antibody levels 35-fold upon receiving a single dose of BNT162b2 mRNA vaccine 15 months after recovery from coronavirus disease 2019 (COVID-19). In contrast, antibody responses in naive individuals boost only 6-fold after a second vaccine. Spike-specific angiotensin-converting enzyme 2 (ACE2) antibody binding responses in the previously infected octogenarians following two vaccine doses exceed those found in a naive cohort after two doses. RNA sequencing (RNA-seq) demonstrates activation of interferon-induced genetic programs, which persist only in the previously infected. A preferential increase of specific immunoglobulin G heavy chain variable (IGHV) clonal transcripts that are the basis of neutralizing antibodies is observed only in the previously infected nuns., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

mRNA-1273 vaccine efficacy against SARS-CoV-2 Delta wanes over time; however, there are limited data on the impact of durability of immune responses on protection. Here, we immunized rhesus macaques and assessed immune responses over 1 year in blood and upper and lower airways. Serum neutralizing titers to Delta were 280 and 34 reciprocal ID 50 at weeks 6 (peak) and 48 (challenge), respectively. Antibody-binding titers also decreased in bronchoalveolar lavage (BAL). Four days after Delta challenge, the virus was unculturable in BAL, and subgenomic RNA declined by ∼3-log 10 compared with control animals. In nasal swabs, sgRNA was reduced by 1-log 10 , and the virus remained culturable. Anamnestic antibodies (590-fold increased titer) but not T cell responses were detected in BAL by day 4 post-challenge. mRNA-1273-mediated protection in the lungs is durable but delayed and potentially dependent on anamnestic antibody responses. Rapid and sustained protection in upper and lower airways may eventually require a boost., Competing Interests: Declaration of interests K.S.C. and B.S.G. are inventors on US patent no. 10,960,070 B2 and International patent application no. WO/2018/081318 entitled “Prefusion Coronavirus Spike Proteins and Their Use”. K.S.C. and B.S.G. are inventors on US patent application no. 62/972,886 entitled “2019-nCoV Vaccine”. L.W., E.S.Y., W.S., J.R.M., M.R., N.J.S. and D.C.D are inventors on US patent application no. 63/147,419 entitled “Antibodies Targeting the Spike Protein of Coronaviruses”. L.P., A. Cook, A.D., A.G., S.K., H.A., and M.G.L. are employees of Bioqual. K.S.C., L.W., W.S., and B.S.G are inventors on multiple US patent applications entitled “Anti-Coronavirus Antibodies and Methods of Use”. A. Carfi and D.K.E. are employees of Moderna. M.S.S. serves on the scientific board of advisors for Moderna. The other authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

Neutralizing antibody responses gradually wane against several variants of concern (VOCs) after vaccination with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine messenger RNA-1273 (mRNA-1273). We evaluated the immune responses in nonhuman primates that received a primary vaccination series of mRNA-1273 and were boosted about 6 months later with either homologous mRNA-1273 or heterologous mRNA-1273.β, which encompasses the spike sequence of the B.1.351 Beta variant. After boost, animals had increased neutralizing antibody responses across all VOCs, which was sustained for at least 8 weeks after boost. Nine weeks after boost, animals were challenged with the SARS-CoV-2 Beta variant. Viral replication was low to undetectable in bronchoalveolar lavage and significantly reduced in nasal swabs in all boosted animals, suggesting that booster vaccinations may be required to sustain immunity and protection.

Current coronavirus (CoV) vaccines primarily target immunodominant epitopes in the S1 subunit, which are poorly conserved and susceptible to escape mutations, thus threatening vaccine efficacy. Here, we use structure-guided protein engineering to remove the S1 subunit from the Middle East respiratory syndrome (MERS)-CoV spike (S) glycoprotein and develop stabilized stem (SS) antigens. Vaccination with MERS SS elicits cross-reactive β-CoV antibody responses and protects mice against lethal MERS-CoV challenge. High-throughput screening of antibody-secreting cells from MERS SS-immunized mice led to the discovery of a panel of cross-reactive monoclonal antibodies. Among them, antibody IgG22 binds with high affinity to both MERS-CoV and severe acute respiratory syndrome (SARS)-CoV-2 S proteins, and a combination of electron microscopy and crystal structures localizes the epitope to a conserved coiled-coil region in the S2 subunit. Passive transfer of IgG22 protects mice against both MERS-CoV and SARS-CoV-2 challenge. Collectively, these results provide a proof of principle for cross-reactive CoV antibodies and inform the development of pan-CoV vaccines and therapeutic antibodies., Competing Interests: Declaration of interests C-L.H. and J.S.M. are inventors on U.S. patent application no. 63/188,813 (“Stabilized S2 Beta-coronavirus Antigens”). This research was, in part, funded by the U.S. Government. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of the U.S. Government. S.R.L., K.H.D., and R.S.B. have a pending patent for recombinant SARS-CoV-2 MA10 used in this study., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

mRNA-1273 vaccine efficacy against SARS-CoV-2 Delta wanes over time; however, there are limited data on the impact of durability of immune responses on protection. We immunized rhesus macaques at weeks 0 and 4 and assessed immune responses over one year in blood, upper and lower airways. Serum neutralizing titers to Delta were 280 and 34 reciprocal ID 50 at weeks 6 (peak) and 48 (challenge), respectively. Antibody binding titers also decreased in bronchoalveolar lavage (BAL). Four days after challenge, virus was unculturable in BAL and subgenomic RNA declined ∼3-log 10 compared to control animals. In nasal swabs, sgRNA declined 1-log 10 and virus remained culturable. Anamnestic antibody responses (590-fold increase) but not T cell responses were detected in BAL by day 4 post-challenge. mRNA-1273-mediated protection in the lungs is durable but delayed and potentially dependent on anamnestic antibody responses. Rapid and sustained protection in upper and lower airways may eventually require a boost.

B.1.351 is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant most resistant to antibody neutralization. We demonstrate how the dose and number of immunizations influence protection. Nonhuman primates received two doses of 30 or 100 µg of Moderna's mRNA-1273 vaccine, a single immunization of 30 µg, or no vaccine. Two doses of 100 µg of mRNA-1273 induced 50% inhibitory reciprocal serum dilution neutralizing antibody titers against live SARS-CoV-2 p.Asp614Gly and B.1.351 of 3,300 and 240, respectively. Higher neutralizing responses against B.1.617.2 were also observed after two doses compared to a single dose. After challenge with B.1.351, there was ~4- to 5-log 10 reduction of viral subgenomic RNA and low to undetectable replication in bronchoalveolar lavages in the two-dose vaccine groups, with a 1-log 10 reduction in nasal swabs in the 100-µg group. These data establish that a two-dose regimen of mRNA-1273 will be critical for providing upper and lower airway protection against major variants of concern., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Immune correlates of protection can be used as surrogate endpoints for vaccine efficacy. Here, nonhuman primates (NHPs) received either no vaccine or doses ranging from 0.3 to 100 μg of the mRNA-1273 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine. mRNA-1273 vaccination elicited circulating and mucosal antibody responses in a dose-dependent manner. Viral replication was significantly reduced in bronchoalveolar lavages and nasal swabs after SARS-CoV-2 challenge in vaccinated animals and most strongly correlated with levels of anti–S antibody and neutralizing activity. Lower antibody levels were needed for reduction of viral replication in the lower airway than in the upper airway. Passive transfer of mRNA-1273–induced immunoglobulin G to naïve hamsters was sufficient to mediate protection. Thus, mRNA-1273 vaccine–induced humoral immune responses are a mechanistic correlate of protection against SARS-CoV-2 in NHPs.

Adjuvanted soluble protein vaccines have been used extensively in humans for protection against various viral infections based on their robust induction of antibody responses. Here, soluble prefusion-stabilized spike protein trimers (preS dTM) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were formulated with the adjuvant AS03 and administered twice to nonhuman primates (NHPs). Binding and functional neutralization assays and systems serology revealed that the vaccinated NHP developed AS03-dependent multifunctional humoral responses that targeted distinct domains of the spike protein and bound to a variety of Fc receptors mediating immune cell effector functions in vitro. The neutralizing 50% inhibitory concentration titers for pseudovirus and live SARS-CoV-2 were higher than titers for a panel of human convalescent serum samples. NHPs were challenged intranasally and intratracheally with a high dose (3 × 10 6 plaque forming units) of SARS-CoV-2 (USA-WA1/2020 isolate). Two days after challenge, vaccinated NHPs showed rapid control of viral replication in both the upper and lower airways. Vaccinated NHPs also had increased spike protein-specific immunoglobulin G (IgG) antibody responses in the lung as early as 2 days after challenge. Moreover, passive transfer of vaccine-induced IgG to hamsters mediated protection from subsequent SARS-CoV-2 challenge. These data show that antibodies induced by the AS03-adjuvanted preS dTM vaccine were sufficient to mediate protection against SARS-CoV-2 in NHPs and that rapid anamnestic antibody responses in the lung may be a key mechanism for protection., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Neutralizing antibody responses gradually wane after vaccination with mRNA-1273 against several variants of concern (VOC), and additional boost vaccinations may be required to sustain immunity and protection. Here, we evaluated the immune responses in nonhuman primates that received 100 µg of mRNA-1273 vaccine at 0 and 4 weeks and were boosted at week 29 with mRNA-1273 (homologous) or mRNA-1273.β (heterologous), which encompasses the spike sequence of the B.1.351 (beta or β) variant. Reciprocal ID 50 pseudovirus neutralizing antibody geometric mean titers (GMT) against live SARS-CoV-2 D614G and the β variant, were 4700 and 765, respectively, at week 6, the peak of primary response, and 644 and 553, respectively, at a 5-month post-vaccination memory time point. Two weeks following homologous or heterologous boost β-specific reciprocal ID 50 GMT were 5000 and 3000, respectively. At week 38, animals were challenged in the upper and lower airway with the β variant. Two days post-challenge, viral replication was low to undetectable in both BAL and nasal swabs in most of the boosted animals. These data show that boosting with the homologous mRNA-1273 vaccine six months after primary immunization provides up to a 20-fold increase in neutralizing antibody responses across all VOC, which may be required to sustain high-level protection against severe disease, especially for at-risk populations., One-Sentence Summary: mRNA-1273 boosted nonhuman primates have increased immune responses and are protected against SARS-CoV-2 beta infection.

Background: Vaccine efficacy against the B.1.351 variant following mRNA-1273 vaccination in humans has not been determined. Nonhuman primates (NHP) are a useful model for demonstrating whether mRNA-1273 mediates protection against B.1.351., Methods: Nonhuman primates received 30 or 100 µg of mRNA-1273 as a prime-boost vaccine at 0 and 4 weeks, a single immunization of 30 µg at week 0, or no vaccine. Antibody and T cell responses were assessed in blood, bronchioalveolar lavages (BAL), and nasal washes. Viral replication in BAL and nasal swabs were determined by qRT-PCR for sgRNA, and histopathology and viral antigen quantification were performed on lung tissue post-challenge., Results: Eight weeks post-boost, 100 µg x2 of mRNA-1273 induced reciprocal ID 50 neutralizing geometric mean titers against live SARS-CoV-2 D614G and B.1.351 of 3300 and 240, respectively, and 430 and 84 for the 30 µg x2 group. There were no detectable neutralizing antibodies against B.1351 after the single immunization of 30 µg. On day 2 following B.1.351 challenge, sgRNA in BAL was undetectable in 6 of 8 NHP that received 100 µg x2 of mRNA-1273, and there was a ∼2-log reduction in sgRNA in NHP that received two doses of 30 µg compared to controls. In nasal swabs, there was a 1-log 10 reduction observed in the 100 µg x2 group. There was limited inflammation or viral antigen in lungs of vaccinated NHP post-challenge., Conclusions: Immunization with two doses of mRNA-1273 achieves effective immunity that rapidly controls lower and upper airway viral replication against the B.1.351 variant in NHP.

Immune correlates of protection can be used as surrogate endpoints for vaccine efficacy. The nonhuman primate (NHP) model of SARS-CoV-2 infection replicates key features of human infection and may be used to define immune correlates of protection following vaccination. Here, NHP received either no vaccine or doses ranging from 0.3 - 100 μg of mRNA-1273, a mRNA vaccine encoding the prefusion-stabilized SARS-CoV-2 spike (S-2P) protein encapsulated in a lipid nanoparticle. mRNA-1273 vaccination elicited robust circulating and mucosal antibody responses in a dose-dependent manner. Viral replication was significantly reduced in bronchoalveolar lavages and nasal swabs following SARS-CoV-2 challenge in vaccinated animals and was most strongly correlated with levels of anti-S antibody binding and neutralizing activity. Consistent with antibodies being a correlate of protection, passive transfer of vaccine-induced IgG to naïve hamsters was sufficient to mediate protection. Taken together, these data show that mRNA-1273 vaccine-induced humoral immune responses are a mechanistic correlate of protection against SARS-CoV-2 infection in NHP., One-Sentence Summary: mRNA-1273 vaccine-induced antibody responses are a mechanistic correlate of protection against SARS-CoV-2 infection in NHP.

Adjuvanted soluble protein vaccines have been used extensively in humans for protection against various viral infections based on their robust induction of antibody responses. Here, soluble prefusion-stabilized spike trimers (preS dTM) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) were formulated with the adjuvant AS03 and administered twice to nonhuman primates (NHP). Binding and functional neutralization assays and systems serology revealed that NHP developed AS03-dependent multi-functional humoral responses that targeted multiple spike domains and bound to a variety of antibody F C receptors mediating effector functions in vitro . Pseudovirus and live virus neutralizing IC 50 titers were on average greater than 1000 and significantly higher than a panel of human convalescent sera. NHP were challenged intranasally and intratracheally with a high dose (3×10 6 PFU) of SARS-CoV-2 (USA-WA1/2020 isolate). Two days post-challenge, vaccinated NHP showed rapid control of viral replication in both the upper and lower airways. Notably, vaccinated NHP also had increased spike-specific IgG antibody responses in the lung as early as 2 days post challenge. Moreover, vaccine-induced IgG mediated protection from SARS-CoV-2 challenge following passive transfer to hamsters. These data show that antibodies induced by the AS03-adjuvanted preS dTM vaccine are sufficient to mediate protection against SARS-CoV-2 and support the evaluation of this vaccine in human clinical trials., Competing Interests: Declaration of interests All authors have declared the following interests: VL, TB, CB, SR, TMF, DC, RC are Sanofi Pasteur employees and may hold stock. MK, RvdM, and CG are employees of the GSK group of companies and report ownership of GSK shares.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative infection of a global pandemic that has led to more than 2 million deaths worldwide. The Moderna mRNA-1273 vaccine has demonstrated ~94% efficacy in a Phase 3 study and has been approved under Emergency Use Authorization. The emergence of SARS-CoV-2 variants with mutations in the spike protein, most recently circulating isolates from the United Kingdom (B.1.1.7) and Republic of South Africa (B.1.351), has led to lower neutralization from convalescent serum by pseudovirus neutralization (PsVN) assays and resistance to certain monoclonal antibodies. Here, using two orthogonal VSV and lentivirus PsVN assays expressing spike variants of 20E (EU1), 20A.EU2, D614G-N439, mink cluster 5, B.1.1.7, and B.1.351 variants, we assessed the neutralizing capacity of sera from human subjects or non-human primates (NHPs) that received mRNA-1273. No significant impact on neutralization against the B.1.1.7 variant was detected in either case, however reduced neutralization was measured against the mutations present in B.1.351. Geometric mean titer (GMT) of human sera from clinical trial participants in VSV PsVN assay using D614G spike was 1/1852. VSV pseudoviruses with spike containing K417N-E484K-N501Y-D614G and full B.1.351 mutations resulted in 2.7 and 6.4-fold GMT reduction, respectively, when compared to the D614G VSV pseudovirus. Importantly, the VSV PsVN GMT of these human sera to the full B.1.351 spike variant was still 1/290, with all evaluated sera able to fully neutralize. Similarly, sera from NHPs immunized with 30 or 100μg of mRNA-1273 had VSV PsVN GMTs of ~ 1/323 or 1/404, respectively, against the full B.1.351 spike variant with a ~ 5 to 10-fold reduction compared to D614G. Individual mutations that are characteristic of the B.1.1.7 and B.1.351 variants had a similar impact on neutralization when tested in VSV or in lentivirus PsVN assays. Despite the observed decreases, the GMT of VSV PsVN titers in human vaccinee sera against the B.1.351 variant remained at ~1/300. Taken together these data demonstrate reduced but still significant neutralization against the full B.1.351 variant following mRNA-1273 vaccination.

Background: Vaccines to prevent coronavirus disease 2019 (Covid-19) are urgently needed. The effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines on viral replication in both upper and lower airways is important to evaluate in nonhuman primates., Methods: Nonhuman primates received 10 or 100 μg of mRNA-1273, a vaccine encoding the prefusion-stabilized spike protein of SARS-CoV-2, or no vaccine. Antibody and T-cell responses were assessed before upper- and lower-airway challenge with SARS-CoV-2. Active viral replication and viral genomes in bronchoalveolar-lavage (BAL) fluid and nasal swab specimens were assessed by polymerase chain reaction, and histopathological analysis and viral quantification were performed on lung-tissue specimens., Results: The mRNA-1273 vaccine candidate induced antibody levels exceeding those in human convalescent-phase serum, with live-virus reciprocal 50% inhibitory dilution (ID 50 ) geometric mean titers of 501 in the 10-μg dose group and 3481 in the 100-μg dose group. Vaccination induced type 1 helper T-cell (Th1)-biased CD4 T-cell responses and low or undetectable Th2 or CD8 T-cell responses. Viral replication was not detectable in BAL fluid by day 2 after challenge in seven of eight animals in both vaccinated groups. No viral replication was detectable in the nose of any of the eight animals in the 100-μg dose group by day 2 after challenge, and limited inflammation or detectable viral genome or antigen was noted in lungs of animals in either vaccine group., Conclusions: Vaccination of nonhuman primates with mRNA-1273 induced robust SARS-CoV-2 neutralizing activity, rapid protection in the upper and lower airways, and no pathologic changes in the lung. (Funded by the National Institutes of Health and others.)., (Copyright © 2020 Massachusetts Medical Society.)