Human Papillomavirus Type 16 L1/L2 VLP Experimental Internalization by Human Peripheral Blood Leukocytes
bioRxiv(2020)
摘要
Human papillomavirus (HPV) accounts for hundreds of thousands of new cases of cervical cancer yearly, and half of these women die of this neoplasia. This study investigates the possibility of HPV16 L1/L2VLP to be internalized by human peripheral blood leukocytes in ex vivo assays. We have developed a leukocyte separation method from heparinized blood samples aiming cellular integrity and viability. We have expressed humanized L1 and L2 viral capsid proteins in HEK293T epithelial human cells, transiently transfecting them with vectors encoding humanized HPV16 L1 and L2 genes. Recombinant L1/L2 capsid proteins and structured virus-like particles interacted with human peripheral blood mononuclear cells, lymphocytes and monocytes, and were internalized through a pathway involving CD71 transferrin receptors. This was observed, at a percentile of about 54% T- CD4, 47% T-CD8, 48% B-CD20, and 23% for monocytes-CD14. The group of polymorph nuclear cells: neutrophils-eosinophils-basophils group did not internalize any VLPs. Blockage assays with biochemical inhibitors of distinct pathways, like chlorpromazine, rCTB, filipin, nystatin, liquemin, and sodium azide also evidentiated the occurrence of virus-like particles indiscriminate entrance via membrane receptor on mononuclear cells. This study shows that HPV16 L1/L2 VLPs can interact with the plasma membrane surface and successfully enter lymphocytes without requiring a specific receptor.
Legend of the Graphical Abstract Graphical abstract showing ex vivo and in vitro internalization between VLPs and host cells.
After leukocytes separation from human whole blood, it was performed the identification of human peripheral blood leukocytes in ex vivo interactions with VLPs. The graph shows that of the cells that interacted with VLPs, 52% corresponded to lymphocytes T-CD4, 47% lymphocytes T-CD8, 48% lymphocytes B-CD20, and only 23% of the monocytes CD14 interacted with these particles. However, monocytes apparently internalized larger amounts of particles when compared to lymphocytes.
It is probable that in some T lymphocytes the amount of internalized particles has been imperceptible to the confocal microscope, since the VLPs produced in this research are around 50 nm in diameter. These results lead to two important implications. First, the interaction of VLPs with lymphocytes may result in the activation of these cells and, consequently, increase the population of these circulating cells, this being crucial in the induction of specific immune response.
In the second implication, these lymphocytes would internalize small amounts of virus, insufficient to activate the immune system. Here it is important to note that lymphocytes are cells capable of dividing and it is estimated that the half-life of these inactive cells in humans is of some years. In addition, as it is known, inactive lymphocytes continually re-circulate through the bloodstream and lymphatic vessels.
The percentage of cells that interacted with the HPV16 L1/L2 VLPs was calculated by the number of cells recognized by the anti-CD antibodies, which internalized these particles. The result corresponds to the analysis in duplicates, being representative of at least four tests.
All images are original and cells were processed by Cianciarullo AM et al., at the Butantan Institute, Sao Paulo – SP, Brazil.
Electron micrographs of human leukocytes, HEK293T and HPV16 L1/L2 VLPs were obtained in a Zeiss EM109 transmission electron microscope. The blue color of the VLPs and colored leukocytes were virtually attributed. Leukocyte and HEK293T present filamentous actin (red) and HPV16 L1/L2 VLPs (green), by fluorescence in a Confocal Zeiss LSM 510 Meta Microscope
![Figure][1]
### Competing Interest Statement
The authors have declared no competing interest.
* HPV
: human papillomavirus
VLP
: virus-like particles
L1
: major capsid protein
L2
: minor capsid protein
HEK293T
: human epithelial cell lineage 293T
CD71
: transferrin receptor
CD4
: T lymphocyte membrane receptor
CD8
: T lymphocyte membrane receptor
CD20
: B lymphocyte membrane receptor
CD14
: monocyte membrane receptor
rCTB
: recombinant cholera toxin B subunit is the non-toxic portion of cholera toxin (CT)
HIV
: human immunodeficiency virus
pUF3L1h vector
: humanized expression vector for L1 protein
pUF3L2h vector
: humanized expression vector for L2 protein
EDTA
: anticoagulant
Tf
: transferrin
DMEM
: Dulbecco’s Modified Medium
FBS
: fetal bovine serum
RPMI
: Roswell Park Memorial Institute cell culture medium
PBMC
: peripheral blood mononuclear cells
BSA
: bovine serum albumin
TEM
: transmission electron microscopy
[1]: pending:yes
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关键词
HPV16 L1/L2 VLP,HPV internalisation by lymphocytes,HPV membrane receptors,cancer risk factor,public health
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