Prediction of effective humoral response to SARS-CoV-2 vaccines in healthy subjects by cortical thickness of post-vaccination reactive lymphadenopathy

Coronavirus disease-19 (COVID-19) is an ongoing global pandemic caused by the 2019 novel coronavirus (2019-nCoV), also known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It was first detected in the Chinese city of Wuhan in early December 2019, after the notification of numerous cases of atypical pneumonia. Only 1 month later, COVID-19 spread quickly worldwide, and the World Health Organization (WHO) declared it a public health emergency of international concern (PHEIC). On March 2020, it was declared a global pandemic. In consequence, multiple public health interventions were implemented worldwide to decrease the transmission of the virus and contain its spread.

After more than 2 years of the COVID-19 outbreak, the only measure guaranteeing virus control has been the implementation of broad vaccination programs. Different types of vaccines have obtained emergency use authorizations (EUA) from the US Food and Drug Administration (FDA) and the European Medical Agency (EMA), including novel mRNA (Pfizer-BioNTech, Moderna) and viral vector-based (AstraZeneca, Janssen) vaccines. Additionally, in June 2021, the WHO's Strategic Advisory Group of Experts on Vaccines approved “mix-and-match” or heterologous COVID-19 vaccine protocols with an initial dose of AstraZeneca and a booster of Pfizer vaccine.

All COVID-19 vaccine platforms are administered intramuscularly but their mechanism of action differs: mRNA vaccines use the virus’s genetic material (RNA) during inoculation while viral vectors used modified versions of a different virus [1]. Their subsequent immune response is a complicated process necessitating several steps, many of which involve the lymphatic system [2]. Vaccine antigens are taken up by antigen-presenting cells (APCs) from the injection site to regional secondary lymphoid tissues, namely the lymph nodes, via lymphatic channels. Lymph nodes represent the activation center of the immune response, where vaccine-peptide antigens are presented by APCs to residing lymphocytes in order to elicit two main responses: cellular response, with the formation of cytotoxic T lymphocytes capable of directly killing infected cells, and a humoral response, which depends on B cells proliferation in the lymph node’s germinal center (GC), resulting in the formation of matured memory B-cells and antibody-secreting long-lived plasma cells [3].

Therefore, widespread cases of reactive unilateral axillary lymphadenopathy were reported after the introduction of the COVID-19 mass vaccination campaign [4‐12]. While axillary and supraclavicular adenopathy ipsilateral to the injection site has also been documented after administration of other vaccines in the past [13‐17], no data exist on the correlation between vaccination-associated reactive lymphadenopathy and the elicited immune response to anti-SARS-CoV-2 vaccines. The aim of this study is to assess, in healthy adults, a potential association between the phenomenon of reactive hyperplastic lymphadenopathy identified on post-vaccination ultrasound and the humoral immunity revealed by serologic testing.

Ever since the rollout of vaccination programs to the entire population, widespread cases of hyperplastic lymph nodes have been reported as ipsilateral to the injection site. However, the association between lymphadenopathy induced by COVID-19 vaccinations and immune response elicited remains unknown. This is the first prospective study that examined the association between cortical morphologic features of reactive axillary lymph nodes and SARS-CoV-2 antibody levels in healthy patients, adding new insights into this clinical pandemic-era conundrum.

All patients recruited in our prospective cohort were employees from our center and, after requesting their written consent, we were allowed to access their personal medical history. No rheumatological or oncological diseases were present at the time of their inclusion in the study or during their follow-up period, and they were therefore considered healthy volunteers. In the study, women represented the largest percentage of the sample (92%) due to a higher proportion of female employees in the health care and social sector [22]. The mean age of recruited patients was similar to the mean age of the population in our country (44 years old) [23].

Ultrasound examination was obtained from the axillary region ipsilateral to the vaccine injection, including the three axillary levels. In our experience, the most frequently hyperplastic reported lymph nodes were located below the lower edge of the pectoralis minor muscle (level I), so this region was more carefully evaluated. The five ultrasound nodal characteristics described in the design of the study were assessed for each visible node in all volunteers. However, the nodal feature chosen to establish an association with antibody levels was the maximum value detected of cortical thickness. This variable is the most relevant clinical-radiological factor, and it is a quantitative measurement with more objectivity and reproducibility as well as an easier statistical assessment. So, regardless of whether a lymph node presented a diffuse and symmetric cortical thickness greater than or equal to 3 mm (type Bedi 3), a generalized lobulated cortical thickening (type Bedi 4), or a focal cortical lobulation (type Bedi 5), we chose its maximum cortical value (measured in millimeters) (Fig. 3).

Before studying the association between the maximum cortical thickness of reactive lymphadenopathy and the quantification of total antibodies, we avoided the possible effect of a previous history of COVID-19 disease in the humoral immune response by dividing the sample into two subgroups: coronavirus-naïve volunteers and previously infected patients. We had two strategies to detect incident cases of COVID-19 among our participants: one, based on information from their clinical record when results of PCR or antigen detection were available and, the second one, based on serological follow-up every 1–3 months. The sequential post-vaccination serologic tests (PVST) performed included the detection of antibodies against both the spike (Anti-S) and the nucleocapsid (Anti-N). All COVID-19 vaccine platforms induce spike protein-specific neutralizing antibodies (Anti-S). However, antibodies against the nucleocapsid (Anti-N) are only generated by natural infection, so their detection in patients (using a cut-off of 0.150) allowed us to diagnose additional COVID-19 cases not detected by direct testing.

Data analysis confirmed statistically higher SARS-CoV-2 IgG antibody levels in individuals previously diagnosed with COVID-19, regardless of the vaccine type used. When comparing the immune response between different vaccine protocols in both groups, we detected higher levels of antibody levels in patients vaccinated with the mix-and-match protocol. This is true for measurements conducted at 21 days, however, differences between the mRNA protocol and the mix-and-match protocol seem less dramatic at 90 days and at 180 days.

To evaluate the association between post-vaccine lymphadenopathy and the effectiveness of the COVID-19 vaccine over time, we defined lymph nodes with a cortical thickness greater than 3 mm as morphological reactive because, following Bedi’s classification, this value supposes the change between normal lymph node appearance (Bedi’s grade 1 and 2) to a suspicious/reactive lymph node appearance (Bedi’s grade 3, 4, 5 and 6). Normally, this classification is used as a way to objectively classify the appearance of lymph nodes in oncologic staging and the likelihood of malignancy increases with each morphologic type [24]. However, due to the lack of malignant risk factors in our cohort, axillary lymph nodes showing large cortical thickness were regarded as physiologic, reactive post-vaccine lymph nodes.

It was possible to calculate the odds of effective humoral response in the subgroup of coronavirus naïve volunteers and statistically significant odds of effective humoral response were found at 90 and 180 days. The fact that the germinal-center (GC) is located in the nodal cortex [25] could explain why cortical thickness developed in post-vaccine lymphadenitis may be the morphological evidence that suggests a more robust B cell germinal-center (GC) response elicited by COVID-19 vaccines. This feature may also indicate a more effective humoral response and a higher likelihood of antibody production, especially at 90 and 180 days.

Areas under the curve (AUC) in convalescent volunteers suggest that cortical thickness holds no diagnostic capacity in this subgroup. This result could be explained by previously published results, which showed a less significant axillary lymph node response to vaccination in patients who were previously infected by SARS-CoV-2 [26]. Regarding the subgroup of coronavirus-naïve patients, the higher AUC value was obtained by analyzing antibody secretion of coronavirus-naïve volunteers at 180 days with acceptable discrimination (AUC = 0.738), followed by antibody secretion of coronavirus-naïve volunteers at 90 days (AUC = 0.713). In both analyses, the best cut-off point was determined to be at a cortical thickness greater than 3 mm, in line with our previous explanations of cortical thickness associated with reactive post-vaccine lymphadenopathy. According to these results, we can conclude that the presence of draining lymph nodes with cortical thickness greater than 3 mm is useful in detecting the effectiveness of the COVID-19 vaccine over time, especially, at 180 days.

The findings in this report are subject to some limitations. Firstly, the immunity to SARS-CoV-2 is likely both humoral and cellular, and this second aspect of response one was not analyzed. Secondly, the selection of healthy employees from our center as the target population eliminated the possibility to report nodal feature changes in extreme ages (sample selection bias) and in individuals with immunocompromising conditions. Thirdly, the small number of volunteers receiving each vaccine protocol does not allow investigating by subclasses. Furthermore, we did not study other nodal locations. Finally, the absence of a baseline axillary ultrasound of vaccine recipients prior to vaccination eliminated the possibility in this study of reporting an induced cortical thickness from a baseline. On the other hand, the scientific rigor of these results is enhanced by its prospective design and the participants’ very high adherence to serial serologic testing.

In conclusion, the key findings of our study include (1) the positive association between the cortical thickness of draining lymph nodes after vaccination and protective antibody titers against SARS-CoV-2 in coronavirus-naïve patients (especially in the long term), and (2) an adequate discrimination capacity of a cortical thickness greater than 3 mm in predicting post-vaccination effective humoral response. These findings provide new insights into previous publications on the relationship between ultrasound features of post-vaccination lymphadenopathy in the context of COVID-19 and inspire further investigations.