Abstract(resumo):

Objective: To compare the percentage of antibodies with neutralizing potential to Wuhan and Omicron variants in the milk and blood of lactating women immunized for SARS-CoV-2 based on the number of doses and vaccine technologies used. Methods: Cross-sectional study conducted in 2022 with 214 lactating women vaccinated for COVID-19 with mRNA, non-replicating viral vector or inactivated whole virus vaccines. Data gathered via standardized questionnaire, and samples of breast milk and blood. Antibodies detected by the ELISA method. A significance level of 5% was considered. Results: Blood had higher neutralizing antibodies percentage than milk for Wuhan (blood-91·3%/milk-23·1%) and Omicron (blood-83·7%/milk-15·2%) variants. Women receiving three or more doses had higher blood antibody percentages for Wuhan (median=91·9%) and Omicron (median=86·0%) variants. In milk, no disparity in the percentage of antibodies was found, but the probability of antibodies' presence was higher than in blood for both variants. Vaccine technologies led to significant differences in the presence of antibodies in milk but not in blood over time for Omicron variant. No difference was observed for Wuhan variant. Conclusion: Protection conferred by antibodies present in milk lasted longer than in blood.

Keywords(palavra-chave):

COVID-19, Human milk, Vaccine, Antibodies, Neutralizing antibodies.

Content(conteúdo):

INTRODUCTION

Due to the rapid increase in the number of confirmed cases and deaths worldwide caused by SARS-CoV-21, several vaccines were developed from different technologies2 and tested in clinical trials3. Four vaccines were approved by The Brazilian Health Regulatory Agency (ANVISA) for use in Brazil: AstraZeneca, Janssen-Johnson & Johnson (non-replicating viral vector), SinoVac/CoronaVac (inactivated whole SARS-CoV-2 virus) and Pfizer/BioNTech (mRNA) 4,5. Although pregnant and lactating women were not included in the vaccine clinical trials, the American College of Obstetricians and Gynecologists and the Brazilian Ministry of Health recommended vaccination of this group6,7 based on articles confirming the safety of the vaccine for both mothers and their infants8,9,10.
Given the importance of breast milk for the immune system of infants, studies examining the impact of SARS-CoV-2 vaccination on breast milk and blood antibodies highlight their importance for infant immune systems9,10. Perl et al.10 found significant IgA and IgG antibodies specific to SARS-CoV-2 in breast milk post-mRNA vaccination, with strong neutralizing effects, potentially safeguarding infants. Similarly, Gray et al9. observed the presence of IgG, IgA, and IgM in the serum of vaccinated women, with antibodies detected in the breast milk of all lactating participants.
Several studies from different countries reported increased neutralizing antibodies against SARS-CoV-2 in human milk from both vaccination and previous infection with the virus. Some of them compared different vaccine technologies11,12,13, others evaluated only mRNA vaccines8,10,14,15. There are also some studies that compared the presence of neutralizing antibodies in human milk and blood of breastfeeding women vaccinated for COVID-19 (with or without previous infection), considering only mRNA vaccines9,16,17,18 or vaccines of different technologies19,20,21, however, there are, so far, no studies that have focused on the analysis of neutralizing antibodies from vaccination based on different variants of the virus, especially in Brazil.
This study aimed to compare the percentage of antibodies with neutralizing potential in the milk and blood of lactating mothers vaccinated against SARS-CoV-2, specifically focusing on the Wuhan and Omicron variants, based on the number of doses and vaccine technologies used. This is crucial for understanding passive immunity in infants, especially since COVID-19 vaccination is not yet available for those under six months old in Brazil22, and the global health impact of COVID-19 persists.

METHODS
STUDY DESIGN, DATA, AND PARTICIPANTS
This cross-sectional study was widely disseminated among donors and patients attending the childcare consultations at the Human Milk Bank of National Institute of Women`s, Children`s and Adolescents` Health Fernandes Figueira (HMB-IFF/Fiocruz-RJ) and among breastfeeding mothers outside the IFF contacted through social networks with information about the eligibility criteria to participate in the study, stages and period of research and contact details for scheduling, carried out via online contact methods.
This study included women who were breastfeeding (exclusively or not), aged 18 years or older, residing in Rio de Janeiro, having completed primary vaccination course with immunizers against COVID-19 available in Brazil, with or without booster doses. Exclusions comprised women with a milk volume below 10 ml and those with less than 5 ml blood volume.
PROCEDURES
For the data collection, a standardized and structured questionnaire tailored for this research and previously tested in a pilot study was used. This instrument administered through “Google Forms” by trained researchers included sociodemographic details, prenatal and clinical information, nutritional status, vaccination dates (first, second, and booster doses), and vaccines manufacturers. The data were collected from April to October 2022.
Milk collection took place at the HMB-IFF/Fiocruz-RJ and was performed by previously trained researchers. A Medela electric pump was used to collect 10 ml of milk. The milk samples were delivered to the High Complexity Laboratory of IFF (LACIFF/Fiocruz-RJ), where they were centrifuged at 2.000 revolutions per minute (rpm) at 4 °C for 25 minutes, after which the supernatants were aliquoted into cryogenic bottles and stored at ?20 °C until use. Before processing, the breast milk samples were thawed and centrifuged at 2.000 rpm for 15 minutes, the fat was removed, and the supernatant was transferred to a new tube. Centrifugation was repeated twice to ensure removal of all fat cells.
The samples of blood were collected by trained professionals at the Nutrition Laboratory of IFF/Fiocruz-RJ. After collection, the samples were delivered to LACIFF/Fiocruz-RJ, where the blood was centrifuged at 3.000 rpm for 10 minutes at room temperature, and the sera were aliquoted into 2 ml Eppendorf tubes and stored at -80 °C until use.
The tests were performed at the Laboratory of Biotechnology and Physiology of Viral Infections, Oswaldo Cruz Institute, Fiocruz-RJ (IOC/LABIFIV). To detect the percentage of antibodies with neutralizing potential for SARS-CoV-2 in the blood and breast milk samples, the enzyme immunoassay (ELISA) method was used with the cPass™ SARS-CoV-2 Neutralization Antibody Kit (GeneScript). This test was designed to mimic the interaction of the virus with the host by direct protein?protein interaction in a direct enzyme immunoassay (ELISA) plate using the purified receptor binding domain (RBD) of the spike protein and the host cell receptor ACE2. Neutralizing antibodies in serum or milk can inhibit this interaction, as in a standard virus neutralization test. This test guarantees 100% specificity and 93% sensitivity for samples above the 30% inhibition cutoff (GenScript).
Concentrations were determined following the manufacturer’s instructions. Samples were diluted in kit-provided buffer in 96-well plates to a final volume of 100 µl. Dilutions (10, 20, 50, or 100 times) were tailored to individual sample requirements. Next, 100 µl of the solution containing the RBD domain of the original SARS-CoV-2 (Wuhan isolate) or of the Omicron variant (B.1.1.529) conjugated to peroxidase (HRP) were added. The plate was incubated at 37 °C for 30 minutes. Subsequently, 100 µl of each well containing the sample and RBD-HRP were transferred to the test plate containing the human ACE2 molecule. The plates were incubated for 15 minutes at 37 °C, the wells were washed four times with 300 µl of the kit wash solution, then 100 µl of the substrate 5·5?-tetramethylbenzidine (TMB) was added for 15 minutes of incubation in the dark at room temperature. The reaction was stopped by adding 50 µl of stop solution from the kit, and the absorbance was read at 450 nm. The calculation of the proportion of antibodies with neutralizing potential was then performed by the following equation, using the negative control of the kit:

Values less than 30% were considered negative. The cutoff was based on validation panels of sera from COVID-19 patients, in which the presence of neutralizing antibodies was confirmed by the 50% plaque reduction neutralization test (PRNT50) and a panel of negative sera. Results are qualitatively reported as positive or negative for neutralizing antibodies presence.
The PRNT50 method uses active virus and is considered the gold standard for the detection and quantification of neutralizing antibodies that mediate viral inhibition. Compared with those in a non-neutralizing antibody control, virus neutralization titers in this assay were reported to result in a 50% reduction in the number of plaques23,24.
STATISTICAL ANALYSIS
Data storage was performed using Excel spreadsheets extracted from “Google Forms”. Categorical variables are described as absolute frequencies and percentages, while numerical variables are presented as medians and interquartile ranges (IQRs). The paired Wilcoxon test was used to compare the percentage of antibodies with neutralizing potential in milk and blood. The Mann?Whitney test was used to determine whether the percentage of this antibodies varied significantly according to the number of doses received. The Kaplan?Meier curve was used to determine the probability of antibodies’ presence over time using the interval between the last dose of vaccine received and milk/blood collection. The log-rank test was used to determine whether there were differences between the Kaplan?Meier curves according to the material analyzed and vaccine technology. All the statistical analyses were performed using SPSS software, version 22 and R, version 4.0.3, with a significance level of 5% (p < 0·05) as a reference.

RESULTS
The study sample comprised 214 lactating women (Figure 1), 56.1% of whom were 35 years of age or older; most were white (71%), lived with a partner (93.9%), had incomplete/complete higher education (92.1%), and had received prenatal care (99.5%). Regarding the prepregnancy body mass index (BMI), 38.3% of the women were overweight (overweight/obese). Regarding chronic diseases, 98.1% did not have pregestational diabetes mellitus or pregestational systemic arterial hypertension (SAH). When asked about the occurrence of SARS-CoV-2 infection prior to vaccination, 62.1% of the participants confirmed that they had already tested positive for COVID-19 (Table 1).
About forty-seven percent (47.7%) of the women received the first dose of the mRNA vaccine (Pfizer/BioNTech), 33.6% received the non-replicating viral vector vaccine (AstraZeneca or Janssen-Johnson & Johnson), and 18.7% received the inactivated whole virus vaccine (SinoVac/CoronaVac). Regarding the second dose, more than half (58.4%) received the mRNA vaccine (Pfizer/BioNTech), 22.5% received the non-replicating viral vector vaccine (AstraZeneca or Janssen-Johnson & Johnson), and 19.1% received the inactivated whole virus vaccine (SinoVac/CoronaVac). Most lactating women (93.5%) were vaccinated with three or more doses (considering the complete cycle of primary vaccination and booster doses) (Table 1).
The analyses of antibodies in milk and blood were performed in relation to the Wuhan and Omicron variant strains. The percentage of antibodies with neutralizing potential was higher in blood than in milk for both variants: Wuhan – blood (median = 91·3% AIQ [IQR: 70·8-94·5%])/milk (median = 23·1% AIQ [IQR: 12·8-34·0%]) (p < 0·001) (Graph 1a); and Omicron blood (median = 83·7% AIQ [IQR: 51·2-93·8%])/milk (median = 15·2% AIQ [IQR: 10·2-24·2%]) (p < 0·001) (Graph 1b).
No disparity in the percentage of antibodies with neutralizing potential in milk was found between women who received two doses (median = 22·9% IQR [12·8-44·9%]) and those with three or more doses (median = 23·5% IQR [12·8-33·3%]) for the Wuhan variant (p = 0·578) (Graph 1c). For blood parameters, the percentage of antibodies with neutralizing potential was notably higher among women with three or more doses (median = 91·9% IQR [71·5-94·7%]) compared to those with only two doses (median = 77·9% IQR [45·6-92·2%]) (p = 0·018) (Graph 1d).
For the Omicron variant, the percentage of antibodies with neutralizing potential in milk was higher among women with two doses (median = 22·5% IQR [20·1–27·9%]) compared to those with three or more doses (median = 15·1% IQR [10·5–22·8%]) (p = 0·334) (Graph 1e). In blood, the percentage of antibodies with neutralizing potential was notably higher among women with three or more doses (median = 86·0% IQR [52·4-94·0%]) than those with only two doses (median = 63·0% IQR [13·9–79·0%]) (p = 0·010) (Graph 1f).
The probability of antibodies' presence with neutralizing potential was higher in milk for both variants concerning the time between the last dose received and the blood/milk collection. For the Wuhan variant analysis, a high percentage of antibodies was observed in the first 50 days post-vaccination (90% in milk and 80% in blood). However, the probability of antibodies' presence in milk decreased by 50% post-vaccination. After 200 days post-vaccination, successive reductions were observed in the following 20 days, stabilizing the survival rate at 30%. In blood, there was a notable and consistent decline, stabilizing after 200 days of vaccination, with only a 5% probability remaining (Graph 2a). Likewise, when analyzing antibodies to the Omicron variant, a high percentage was noted in the initial 50 days post-vaccination (100% in the milk and 80% in the blood), with a steady decline in the probability of antibodies in blood, reaching 50% after 100 days and dropping to minimal levels (10%) at 200 days post-vaccination. In contrast, milk showed a slight decline over 100 days, remaining stable with 85% probability of antibodies presence until 190 days after vaccination (Graph 2b). In summary, antibodies in milk provided longer-lasting protection compared to blood for both the Wuhan variant (p < 0·001) (Graph 2a) and the Omicron variant (p < 0·001) (Graph 2b).
In the analyses of antibodies against the Wuhan variant, no significant difference was observed in the probability of the presence of antibodies with neutralizing potential over time when different vaccine technologies were analyzed, neither in milk (p = 0·900) (Graph 3a) nor in blood (p = 0·800) (Graph 3b). In milk, a decrease in antibody percentage was observed, dropping to 90% at 15 days, then sharply declining to 75% at 50 days, persisting at this level for the subsequent 200 days with inactivated whole virus technology. With non-replicating viral vector technology, the antibody percentage decreased to about 75% between 50 and 100 days, further declining to 60% by 200 days post-vaccination. Using mRNA technology, the percentage of antibodies with neutralizing potential continuously decreased to 45% after up to 180 days (Graph 3a). In blood, at 50 days, there was a 50% decrease in the percentage of antibodies with neutralizing potential, remaining at 20% after 170 days with inactivated whole virus technology. In non-replicating viral vector technology, this percentage was approximately 55% after 90 days, steadily declining over time, reaching minimum levels (10%) at 200 days. With mRNA analysis, the antibody percentage continuously decreased over 200 days, remaining below 10% at 240 days post-vaccination (Graph 3b).
For the Omicron variant, significant difference was noted in the probability of the presence of antibodies with neutralizing potential over time in milk (p < 0·001) (Graph 3c) but not in blood (p = 0·700) (Graph 3d) when comparing vaccine technologies used. In milk, the percentage of antibodies remained stable at 100% for 40 days, after which it declined to the minimum level (10%) before reaching 50 days post-vaccination via inactivated whole virus technology. In non-replicating viral vector technology, the probability of antibodies remained stable at 100% for about 180 days, before declining to levels below 10%. With mRNA analysis, the antibody percentage decreased subtly, remaining at high levels (90%) for over 200 days post-vaccination (Graph 3c). Regarding blood analysis, with inactivated whole virus technology, a probability of antibody production of approximately 60% at 50 days and 20% at 150 days post-vaccination was observed due to successive drops during this period. With non-replicating viral vector technology, there was an approximately 75% probability of antibodies' presence 50 days after vaccination, declining continuously over the following 200 days to levels below 10%. Similarly, with mRNA technology, an approximately 80% probability of antibodies was observed after 50 days, decreasing to less than 10% at 250 days post-vaccination (Graph 3d).

DISCUSSION
The study found more antibodies with neutralizing potential in blood than milk for Wuhan and Omicron variants. Antibodies' presence varied over time, but milk antibodies provided longer-lasting protection. Despite methodological disparities with Lechosa-Muñiz et al. 25, where all participants were previously uninfected, results were similar. Notably, the current study revealed no significant difference in neutralizing antibody potential in milk or blood concerning prior COVID-19 infection.
Lechosa-Muñiz et al.25 conducted a cross-sectional study on breastfeeding women vaccinated with mRNA and non-replicating viral vectors. They also observed notable differences in serum IgG concentration (p < 0·001) post-vaccination but not in breast milk IgG concentration. Similarly, Charepe et al.16 studied 24 female healthcare professionals, analyzing human milk from 14 participants and serum from the entire sample after COVID-19 vaccination via mRNA technology, three weeks post both first and second doses. Post-second dose, they observed increased IgG and IgM levels and decreased serum IgA. In breast milk, IgG rose while IgA declined after the second dose, with no IgM presence detected at either dose.
Furthermore, the study found that for both Wuhan and Omicron variants, lactating women who received only two doses had a lower percentage of antibodies with neutralizing potential in blood compared to those who received three or more doses of various immunizing agents used in Brazil. Regarding milk, no significant difference was found in the percentage of antibodies to the Wuhan variant; however, for the Omicron variant, a higher percentage of antibodies was observed in the milk of women who received only two doses compared to those vaccinated with three or more doses.
Rick et al.19 conducted a prospective cohort study analyzing breast milk and blood from 45 women before and after the COVID-19 booster dose. Similar to the present study, they observed that 96% of participants had antibodies in blood samples before the booster, with increased quantities two to four weeks post-booster and no reduction during days 60 to 119, remaining consistent with pre-booster levels after 120 days. In breast milk, 53% of samples were antibody-positive pre-booster, with an increase observed between 14- and 35-days post-booster and a continuous rise between days 60 and 119, differing from the present study's milk evaluation results.
The study revealed a lower percentage of antibodies with neutralizing potential for the Omicron variant compared to the Wuhan variant in both milk and blood of lactating women vaccinated with two doses or three or more doses. This could be attributed to the vaccines available in Brazil during the study period not covering the Omicron variant, which was identified in Brazil only in November 202226,27. Furthermore, the Comirnaty Bivalent Pfizer vaccine, recommended for booster doses against SARS-CoV-2 variants including Omicron, was incorporated into the vaccination schedule only in February 202328.
The study showed a high percentage of antibodies with neutralizing potential in both blood and milk during the initial 50 days post-vaccination, gradually declining over time. In a prospective study with 29 lactating women, Kelly et al.14 observed a significant increase in IgA and IgG antibodies in breast milk following vaccination with the Pfizer-BioNTech mRNA immunizing agent. IgG levels remained persistent for 20 to 40 days post-first dose, with a possible gradual decline in IgA over time after the second dose. Baird et al. 15, in a longitudinal cohort study of seven lactating women vaccinated with mRNA technology against COVID-19, observed a rise in IgA and IgG levels on the seventh day post-first dose. There was a significant decline before the second dose, followed by a notable increase post-second dose, sustaining high levels 80 days post-vaccination. These findings underscore the significance of all vaccine doses in maintaining antibodies with neutralizing potential levels in breast milk and blood for the sustained protection of lactating women and their infants.
Regarding the percentage of antibodies with neutralizing potential based on the vaccine technologies used, the study revealed differences in the probability of detecting these antibodies in milk and blood for both analyzed variants. However, there was no significant difference observed for the Wuhan variant.
There are variations in the WHO's recommendations regarding the interval between doses for different SARS-CoV-2 vaccine technologies. For inactivated whole virus technology (SinoVac/CoronaVac), an interval of two to four weeks (14 to 28 days) is recommended29; for non-replicating viral vector technology, the recommended interval is eight to twelve weeks (56 to 84 days) for the AstraZeneca immunizer30 and two to six months (60 to 180 days) for the Janssen-Johnson & Johnson immunizer31 in the case of a single-dose booster. For mRNA technology (Pfizer/BioNTech), the interval between doses should be four to eight weeks (28 to 56 days)32. Regarding booster doses, for priority groups, the recommendation is a minimum interval of four months (120 days) after the last dose received28,33,34. As per recommendations, the present study demonstrated that the decline in the percentage of antibodies with neutralizing potential in both blood and milk aligns with the expected time intervals for all analyzed vaccine technologies, without compromising the immunity of lactating women.
The study revealed a reduction in antibodies with neutralizing potential over time in both milk and blood for the two SARS-CoV-2 variants analyzed, regardless of the vaccine used. Interestingly, inactivated whole virus technology experienced a greater decline in the first 50 to 100 days post-vaccination but maintained higher levels of antibodies against the Wuhan variant compared to other agents for the next 150 to 200 days, especially in milk. Conversely, mRNA technology maintained higher antibody levels than other agents for the initial 150 days but exhibited an exponential decrease, thereafter, reaching lower levels than other technologies.
For the Omicron variant, the non-replicating viral vector and mRNA technologies maintained higher antibody percentages over 200 days after vaccination, especially in milk, than did inactivated whole virus technology, which decreased to minimum levels before 50 days post-vaccination. However, caution is advised in interpreting these results as only three lactating women in this sample received the inactivated whole virus technology for their last dose. Consequently, the Kaplan?Meier curve was based solely on this small number for this vaccine technology, potentially leading to inaccurate results, as this behavior was not observed for the other vaccine technologies analyzed.
Based on this evidence and considering the preferred recommendation for the use of mRNA technology in Brazil for pregnant and breastfeeding women during the data collection period of this study35, the importance of booster doses according to the vaccination schedule was confirmed. Additionally, due to this recommendation, although the number of participants immunized with this technology in this study was higher at all doses than the other vaccine agents used, it was important to evaluate the performance of the other immunizers in the analyzed group.
Although the percentage of antibodies with neutralizing potential in breast milk is lower than that in blood, this study confirms the importance of breast milk for infants as a possible way of acquiring passive immunity, given that vaccination for children younger than six months in Brazil has not yet been implemented22.
Regarding the methodologies used to detect and quantify neutralizing antibodies, most studies on this topic have used Enzyme-linked immunosorbent assays (ELISAs), with various analysis kits employed. While the PRNT50 assay is considered the gold standard, it necessitates an active virus similar to natural infection, making it time-consuming, expensive, and requiring biosafety level 3 (BL3) facilities and specialized personnel, and is subject to biological variability. For these reasons, we chose the ELISA with the cPass™ SARS-CoV-2 Neutralization Antibody Kit (GeneScript), which does not require BL3 facilities, permits the analysis of several samples in less time and offers higher efficiency. Additionally, it quantifies antibodies with neutralizing potential against relevant SARS-CoV-2 antigens, such as protein S and the RBD24.
Unlike other published studies10,15,16,21,25, this study did not compare the different isotypes of antibodies present in breast milk and blood since the objective was to compare the total percentage of antibodies with neutralizing potential based on the variants, number of doses applied and vaccine technologies used, making comparisons between the available findings difficult.
Another notable point is that while the present study was conducted during the circulation of the Wuhan variant B.1.1.28 and B.1.1.3326,36, antibody analysis also considered the Omicron variant (B.1.1.529 – BA.1, BA.1.1, BA.2 lines) due to its high transmissibility26,27,37.
In summary, the importance of the present study is reinforced by the use of data from Brazilian women immunized with ANVISA-approved vaccine technologies, in addition to analyzing antibodies with neutralizing potential for two SARS-CoV-2 variants.

ACKNOWLEDGEMENTS
The authors would like to address their gratitude:
All professionals of the National Institute of Women`s, Children`s and Adolescents` Health Fernandes Figueira (IFF/Fiocruz-RJ) and the Laboratory of Biotechnology and Physiology of Viral Infections, Oswaldo Cruz Institute, Fiocruz-RJ (IOC/LABIFIV) who contributed to the realization of this study.
All volunteer professionals who participated in data collection.
All breastfeeding women who participated in the research.

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