Background

Understanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible.

Methods and findings

To find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10-15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes. Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension.

Conclusions

Leukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes.

Zika virus (ZIKV) infection during pregnancy causes congenital abnormalities, including microcephaly. However, rates vary widely, and the contributing risk factors remain unclear. We examined the serum antibody response to ZIKV and other flaviviruses in Brazilian women giving birth during the 2015-2016 outbreak. Infected pregnancies with intermediate or higher ZIKV antibody enhancement titers were at increased risk to give birth to microcephalic infants compared with those with lower titers (P < 0.0001). Similarly, analysis of ZIKV-infected pregnant macaques revealed that fetal brain damage was more frequent in mothers with higher enhancement titers. Thus, features of the maternal antibodies are associated with and may contribute to the genesis of ZIKV-associated microcephaly.