Essay: What Vaccinations Do

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Abstract
Vaccines need to have high affinity B cells to neutralize and stop the spread of viral infections. Monitoring follicular T cells, as they are key members in vaccination, helps one to predict their ability to elicit humoral responses leading to protection against harmful microbes, and preventing said microbes from replicating. Production of high affinity B cells depend on the B cells’ interaction with follicular T cells in the germinal center. Transcription factor Bcl6 delegates differentiation of Tfh cells, while Tfh cells induce massive B cell: proliferation, differentiation into plasma cells or memory cells, affinity maturation and class switching in the germinal center. Results indicate that, interaction between CD28, expressed on naïve CD4+ and CD8+ T cells, and B7, expressed on antigen presenting cells (APC), are required for germinal center formation. Via DNA priming, Tfh cells are readily differentiated which advertently increases GC response. Creating an immune profile can aid in measuring the effects of vaccination on cytokines/chemokines in PBMC and it has shown that IFN- y is the most secreted cytokine followed by IL-6 and IL-8.

Introduction
The environment contains a variety of infections which range from viruses, fungi, bacterial and parasitic, each causing diseases to their own capability and may at times be fatal if left untreated. The immune system plays a huge role in combating these infectious agents. Because these agents vary, the immune system must produce specific immunity to a single microorganism or a group of similar organisms. B-cells and T-cells, specifically Th17, GC B cells, T follicular regulatory cells (Tfr), follicular dendritic cells (FDC) and follicular T helper cells (Tfh) work coordinately to fight against any pathogen introduced to the human host.
The first vaccine was not introduced until the late 1700s and since then there has been major significant improvement to provide protection against certain infections. Vaccination produces active immunity where the vaccine interacts with the immune system eliciting a similar immune response to that of biological infections. Unlike naturally occurring infections however, they do not produce any symptoms and is not disease-causing (1. The immune system and vaccination | Immunisation Advisory Centre. Immuneorgnz. 2017. Available at: http://www.immune.org.nz/immune-system-and-vaccination. Accessed April 19, 2017.) . Through vaccination, one’s immune system produces an immunological memory that is long-lasting. During active immunity, the immune system generates memory B cells and plasma cells, originating in the germinal center (GC). When they exit the GC, the do not differentiate into plasma cells but remain as migrating memory cells (2. Plotkin S. Vaccines. 1st ed.; 2013.). Upon secondary antigen exposure, memory B cells divide rapidly and differentiate into plasma cells secreting antigen-specific antibody as depicted in the figure below (2. Plotkin S. Vaccines. 1st ed.; 2013.).
Figure 1.
B cells differentiation before leaving the lymph node (Plotkin S. Vaccines 1st ed.; 2013).

There are two main types of T cells: cytotoxic T cells and helper T cells. While effector cytotoxic T cells directly kill infected cells, effector helper T cells help in stimulating the responses of other cells such as B cells, APC and cytotoxic T cells. Before these two cells become effector cells, they must first be activated. This takes place in the peripheral lymphoid organs on the surface of APC with an ingested antigen. When the antigen is moved to the surface of an MHC molecule, FDC presents the foreign antigens to T cells, whether it be cytotoxic T cells (CD8+) or helper T cells (CD4+), which then becomes activated.
There are two different types of MHC molecule: MHC class I which presents antigens to cytotoxic T cells (CD8+) and MHC class II which presents antigens to helper T cells (CD4+) as depicted in figure 2. Both complexes are heterodimers with an extracellular N-terminus end which allows for binding of antigen for presentation to T cells. While MHC class I is composed of four extracellular domain, a1, a2, a3 and B2-microgobulin in which a3 and B2-microgoblulin are Ig-like, in MHC class II, the four extracellular domains are a1, a2, B1 and B2 in which a2 and B2 are Ig-like. In both, at the peptide-binding site is where foreign antigens are expressed to T cells (Figure 3).
Altogether, most vaccines, supported by Tfh cells, provide protection by creating antibodies which prevent the pathogen from becoming infectious while also targeting it for killing. Therefore, Tfh cells play a central role in vaccine-mediated antibody responses. Vaccine-induced antibodies can either elicit the extrafollicular response which produces short-lived plasma cells or act on the GC to elicit long-term protection against specific pathogens. Tfh cells mediate the signals to GC B cells while positive selection of B cells leads to specific B cell responses (figure 4).
The GC is where most of the B cell and T cell interaction takes place and their interaction is critical for GC reaction and production of long-lived memory and plasma cells. In the dark zone of the GC and after immunization, activated B cells migrate to the B cell follicle, proliferate and undergo somatic hypermutation. B cells then travel to the light zone where discriminated Tfh cells reside. Activation of CD4+ T cells occurs in the paracortex of the lymph node after presentation of Ag by FDC. Interaction between CD28/B7, ICOS/ICOS-L and OX40/OX40L on CD4+ T cells and FDC upregulates the expression of Bcl6, a transcriptional repressor and CXCR5, a chemokine receptor, to aid in their migration to the B cell follicle. Before exiting the GC as either plasma cells or memory B cells, B cells that have undergone somatic hypermutation are tested. They collect antigens-containing immune complexes from follicular dendritic cells (FDC) and subsequently present those antigens to Tfh cells on MHC class II. Those that express the most antigen to Tfh cells return to the dark zone to be proliferated. This leads to the generation of high affinity germinal center B cells which later exit the GC as plasma cells or memory cells. High affinity B cells are favored in vaccines to prevent and contain any disease.
Overall, in this T-cell dependent B-cell activation reaction, the antigen, a protein, is first ingested then broken into peptides fragments by an APC. The antigen then migrates to the surface of the APC on an MHC class II. Receptors on the T-cell binds to the peptide on the MHC class II molecule and becomes activated and can now stimulates B cells, after the formation and interaction of a MHC class II- B cell complex interacts with a receptor on the activated Th2 cell, into antibody producing cells and memory cells. This process is mediated by haptens when conjugated to a protein carrier. Hapens are small molecules which induce T-cell dependent immune responses and are not immunogenic by itself. They become immunogenic when paired to a protein carrier. They are recognized by IgG receptor on B cells and are then endocytosed. This more or less causes, as the end-result, the activation of B cells and helper T cells.

Follicular T cells (Tfh) are found in the blood and germinal center of secondary lymphoid organs. Differentiation of Tfh cells from other subsets of CD4 T cells are their surface receptors. Tfh cells express PD-1, CD95, ICOS, CXCR5 which shows the presence of humoral response and trancription factor Bcl-6 which aids in Tfh cells’ differentiation and development. Tfh cells, however rely heavily on Bcl-6, IL-21 and IL-6 for its generation. While the mechanism of this specific subset of CD4 T cells are not fully understood, this paper will examine the role of Tfh in viral infections such as Ebola, SIV/HIV, influenza virus and smallpox in vaccination.

Hypothesis/Objectives

Follicular T cells is vital in aiding B cells to promote protective antibody responses during vaccination. Three vaccines are investigated in this paper: Vaccinia virus (VACV), HIV-1, and Zaire Ebolavirus (ZEBOV) and different techniques were used to measure the response of each vaccine on Tfh cells and GC B cells when induced by said vaccines.
Hollister et al. (2014) aimed to show the advantages of using DNA priming in antibody response. By incorporating a DNA priming step in vaccination, they hope to see an increase in Tfh cells differentiation and an heightened GC response.
In addition, interaction between CD28 and B7 (B7-1 and B7-2) exhibit different biochemical reaction though both ligands are homologous. Using the vaccinia virus infection, Salek-Ardakani et al. (2012) aimed to better differentiate the effect both ligands have on Tfh cell development. More specifically, they hope to show that expression of B7-1 alone is not enough for optimal Tfh cell development, GC B cell differentiation and extrafollicular plasma cell differentiation.
Lastly, Farooq et al. (2016) intended to analyze the effect caused by rVSV-ZEBOV vaccine to circulating Tfh cells and cytokines induced by the vaccination. This aided them in creating a cytokine profile induced by the rVSV-ZEBOV vaccine.

Table:

Study 1
Vaccinia virus (VACV) is DNA virus and successful immunization provides protection against the causative agent of smallpox, variola virus. As was aforementioned, B cells interact with T cells to produce antibodies to target viruses. Specifically, of the two types of B7 surface markers, B7-1 and B7-2 (CD 80 and CD 86 respectively), B7-2 interaction with CD28 is more important in virus-specific neutralizing antibody responses per Salek-Ardakani et al. (2012) (12. Salek-Ardakani S, Choi Y, Rafii-El-Idrissi Benhnia M et al. B Cell-Specific Expression of B7-2 Is Required for Follicular Th Cell Function in Response to Vaccinia Virus. The Journal of Immunology. 2011;186(9):5294-5303. doi:10.4049/jimmunol.1100406.). In VACV, antibody responses are predominantly CD4 T cell-dependent responses (13. Xu R, Johnson A, Liggitt D, Bevan M. Cellular and Humoral Immunity against Vaccinia Virus Infection of Mice. The Journal of Immunology. 2004;172(10):6265-6271. doi:10.4049/jimmunol.172.10.6265. ) meaning, B cells activation requires contact with T cells activated by the same antigen. In general, when the host is immunized or infected with the virus, naïve CD4 T cells is activated by recognition of antigen while naïve B cells encounter the antigen through the interaction with follicular T cells (TFH). Because of this chain reaction, B cells can either differentiate into memory cells by moving into B cells follicles to form germinal centers (GC) or effector cells that are short-lived (14. McHeyzer-Williams L, Pelletier N, Mark L, Fazilleau N, McHeyzer-Williams M. Follicular helper T cells as cognate regulators of B cell immunity. Current Opinion in Immunology. 2009;21(3):266-273. doi:10.1016/j.coi.2009.05.010.
15. McHeyzer-Williams L, McHeyzer-Williams M. ANTIGEN-SPECIFIC MEMORY B CELL DEVELOPMENT. Annual Review of Immunology. 2005;23(1):487-513. doi:10.1146/annurev.immunol.23.021704.115732.
16. Okada T, Cyster J. B cell migration and interactions in the early phase of antibody responses. Current Opinion in Immunology. 2006;18(3):278-285. doi:10.1016/j.coi.2006.02.005.).

Methods
Mice: Eight-to-12-wk-female CD57BL/6, CD28, B7, B2 mice were purchased from The Jackson Laboratory in addition to B7-1 and B7-2 double deficient mice.
Peptides: VAVC peptide epitopes used such as A18R, D13L, E9L and J4R are examples of antigens recognized by VAVC-specific CD4+ T cells and are targeted by CD8+ T cells ( Vaccinia Virus-Specific CD4+ T Cell Responses Target a Set of Antigens Largely Distinct from Those Targeted by CD8+ T Cell Responses).
In vivo Ab treatment: Hydromas were cultured and mAbs (monoclonal antibodies) were isolated via the supernatant. To block the B-cells, anti B7-1 and anti B7-2 of intraperitoneal injection (i.p) were given on days 0-3 days after infection with the VAVC.
VACV preparation: VAVC were prepared using HeLa cells as described by ___________.
Virus Infection: Mice were infected bilaterally with 2.5×105 PFU VAVC- Western type via i.p injection. CD4+ T cells, differentiated memory B cells, Tfh cells and VAVC specific antibody responses were then analyzed on three different occasions post-infection on day 8, 15 and 35.
Flow Cytometry: Cells from the spleen of euthanized mice were analyzed and suspended. Following RBC lysis, cells were then stained with anti-Fc II/III receptor mAB for 15mins at 4C.
Germinal Staining: The germinal center was stained with anti-mouse IgD, B220, PNA and FAS. They were washed again and stained with streptavidin-APC and were then analyzed for surface expression of B7-1 and B7-2. Plasma cells and Tfh cells were stained using anti-mouse CXCR5 followed by anti-rat IgG in PBS. Cytokine production was measured in CD4+T cell followed by the measurement of the level of specific antibodies against VACV in serum. VACV was then neutralized while B cells were isolated from the spleen of euthanized mice in which CD19+ B lymphocytes from naive WT mice were transferred into B7-1-/-, B7-2-/-, CD28-/- or B7-1.2-/-. GC responses and plasma cells differentiation were examined.

Results
There are two types of cells in the lymph node: normal Tfh cells and GC Tfh cells. The result indicated that B7-2 plays a major role in Tfh response to VACV as they are more likely to interact with CD28 in the germinal center in order to develop GC Tfh cells. As stated previously, GC Tfh cells can be distinguished by the expression of CXCR5, PD-1, ICOS molecule, and high expression of Bcl6. Post-infection, antibody specific CD4 cells peaked between day 8 and 10. Upon analysis of cells collected on day 8, the absence of B7-2 caused a decrease in the number of Tfh cells and GC Tfh cells while those variables were not affected B7-1 -/- deficient mice. Furthermore, B7-2 is not only needed for the maturation of GC cell phenotype and plasma cell differentiation in responses to VACV but is also important in class switching and the expression of Bcl6 as discussed earlier in response to any vaccine. By infecting mice with VACV-WR, researchers saw an increase in B cell expression of Bcl6. Lastly, at day 30 post-infection, optimal neutralizing agents against VACV was detected. While most VACV-WR B7-1 -/- infected mice were able to escape death and experienced less symptoms such as weight loss, those deficient in B7-2/CD28 did not face the same fate.

Conclusion
Overall, B7-2 is expressed at a much higher rate in response to VACV which proves a deficiency in B7-2 will have a huge negative impact on Tfh cell development in the GC.

Study 2
An effective vaccine entails the production of high affinity antibodies. While there has been a clearer understanding of the roles played by the GC and Tfh in humoral immune response, little is known about the role of Tfh cells in the HIV-1 vaccine. Most published works have shown the advance of DNA priming in vaccines such as influenza, malaria and tuberculosis (PAPER INTRO 2nd paragraph). For example, in a past research mice immunized via DNA priming with the gp120 version of HIV-1 GP resulted with high affinity and specific antibodies than vaccines alone. Because Tfh cells are pivotal for plasma cells secreting high affinity B cells, Hollister et al. (2014) postulated DNA priming will cause Tfh cells to differentiate at a higher pace in comparison to protein priming.
Methods
Mice and Immunization: Eight to ten-week old mice of both genders were primed with gp120-encoding DNA, a glycoprotein expressed on the surface of HIV cells, and gp 120 protein boost vaccination to generate titers with high affinity. Prior to this, BCL6 mice were mated to UBC-Cre-ERT2 mice where there is a double expression of both a human estrogen and the cre recombinase gene guided by the UBC gene promoter. The pilot study mice were then immunized i.m with sheep red blood cells (SRBC) from a constructed codon optimized JR-FL gp120 DNA vaccine. The mice were immunized i.m with either DNA or gp120 protein in ALUM(AL(OH) 3 and i.p injections of Tamoxifen. Flow Cytometry: Active spleen cells were then incubated with anti-mouse CD16/CD32 followed by surface staining to visualize the mentioned markers. Examination of B cells and T cells reaction and interaction in the spleen were measured using antibodies detailed in Hollister et al. (2014).
B and T cells analysis: Percentage of total Tfh cells and GC B cells were identified in the following manner:
Percentage total of Tfh cells = (% CD4+ CDCR5+ double positive T cells in the spleen) x (% ICOS+ PD-1 double positive cells within the CD4+ CXCR5+ population)
Percentage total of GC B cells= (% of B220+Fas+ double positive B cells within total spleen population) x (% PNA+ GL7+ B cells within the B220+ Fas double positive cells within the B cell population)
Antibody titer analysis: Abs titers go gp120-specific IgG were analyzed using ELISA while Abs avidity was measured via the NASCN displacement method.

Results
Based on Hollister et al. (2014) experiment, four key results were obtained. First, it was noted that immunization with gp-120 encoding DNA elicits a stronger GC response than gp-120 protein itself as indicated in figure 6a.
Three days post final injection, mice immunized with gp-120 encoding DNA had an increase in both, Tfh cells and GC B cells in the spleen though the increase in Tfh cells were not significant as seven days post final injection, it’s number equaled that of the mice primed with gp-120 protein alone. Antibody analysis indicated gp-120 protein, overall, causes a higher secretion of antigen-specific IgG titers (Figure 6C). However, the avidity of gp120-encoding DNA was higher than that of gp120 protein (Figure 6D). All-in-all, compared to its counterpart, gp120- encoding DNA generates a higher GC response, higher antigen-specific IgG post infection/injection and higher Tfh cell response.
Figure 6. gp120-encoding DNA elicit an earlier response post immunization. At day 7, gp 120 protein alone had higher Tfh cell percentage though not significant. GC B cells are significantly induced rapidly and at a higher rate than gp120 protein alone.

Secondly, priming with gp120-encoding DNA improves the quantity of Tfh cells in the spleen while also enhancing the GC. GC B cells were higher up to seven days post final immunization in mice immunized with gp120-encoding DNA while Tfh cells from the spleen decreased by day 7 (Figure 7 ).

Figure 7. Significant increase of GC B cells day 3 and 7 post infection of gp120-encoding DNA versus gp120 protein. Significant increase of Tfh cells day 3 post infection with gp120-encoding DNA at day 3 and insignificant increase at day 7.

Even with boosting, the increase in Tfh cells were not significant and paralleled the number of Tfh cells in mice primed with gp120 protein alone by day 7. This demonstrates benefit of priming with DNA encoded gp120 rather than protein alone, as it increases GC B cell populations and elicit an earlier immune response. In addition, when effector memory CD4+ T cells were examined, no significant differences were noted between day 3 and 7 post final injection. Upon further analysis, it was found that the proportion of Tfh cells, which are activated helper T cells, within effector memory cells were significantly higher three days post gp120-encoding DNA immunization. By day 7, it slightly decreased while gp120 protein increased (Figure 8). This data demonstrates priming with gp120-encoding DNA enhances the secretion of Tfh cell.
Figure 8. Enhanced levels Tfh cells post final injection.
Third, priming with gp120-encoding DNA enhances GC activity earlier than gp120 protein alone. Three days after final immunization, titers of gp120-encoding DNA were higher, as was the avidity. At day seven, the antibody and avidity of mice primed with gp120 protein alone equaled that of those primed with gp120-encoding DNA. Both priming methods however, caused (1) a decrease in memory B cells, (2) B cells to differentiate from memory cells to a transitional state and (3) an increase in transitional B cells from a memory cells to an active cell state.
Lastly, elimination of Bcl6 caused an increase of gp120-specific IgG. By mating Bcl6 knock-out mice to mice expressing a Cre-ERT2 and administering Tamoxifen to these mice, Hollister et al. (2014) were able to study the immune response. Tamoxifen is an estrogen homolog which can cause the translocation of the cre-ER protein into the nucleus where recombination of Bcl6 DNA happens. Essentially, Tamoxifen aids in the deletion of Bcl6. Three days post final immunization, gp120-encoding DNA elicited a higher Tfh and GC B cell production in spleen of mice not treated with Tamoxifen. The population of Tfh cells and GC B cells in mice treated with Tamoxifen were significantly decreased in the spleen. Deletion of Bcl6 caused an increase of antigen-specific IgG and their avidity in treated mice (Figure 9). This only further validates the importance of Bcl6 for GC development.

Figure 9.
Deletion of Bcl6 with Tamoxifen caused a decrease in GC B cells, Tfh Cells and an increase in antigen-specific IgG.

Conclusion
Taken together, Hollister et al. (2014) showed (1) immunization with gp-120 encoding DNA has a greater effect on GC response, antigen-specific IgG and Tfh cell response compared to gp-120 protein itself; (2) priming with gp120-encoding DNA amplifies the secretion of Tfh cells and GC activity at an earlier time point versus gp-120 protein alone; and (3) deletion of Bcl6 induced gp120-specific IgG.

Study 3

Very recently, in 2014, the Ebola outbreak emphasized the critical need for an effective vaccine. Ebolavirus are from the family Filoviridae (filovirus) of the order Mononegavirales. Infections via this virus is highly pathogenic and can be transmitted via bodily fluids from infected host, whether it be human or zoonotic (The lifecycle of the Ebola virus in host cells 1st parargraph intro). The first reported case of Ebola was reported and recognized in 1976 in Zaire, the Democratic Republic of the Congo (DRC) and South Sudan. According to the CDC, the disease was spread by close personal contact and by use of contaminated needles and syringes in hospitals/clinics (https://www.cdc.gov/vhf/ebola/outbreaks/history/chronology.html). Since 1976, scientist has been able to recognize different subsets/species of the Ebola virus, in which their names specify their destination. The five different species are: 1) Zaire virus (Zaire EBOV), 2. Sudan virus (Sudan EBOV), 3. Bundibugyo virus (Bundibugyo EBOV), 4. Tai Forest virus (Tai Forest EBOV) and 5. Reston virus (Reston EBOV). The most recent outbreak in 2014 exceeded in terms of number of reported cases and geographical range in comparison to previous epidemics. From 2014-2017, more than 28,000 human cases of the Ebola virus was reported with a total of more than 11,000 deaths affective multiple countries. While the outbreak originated in Guinea, Liberia and Sierra Leone, due to migration, confirmed cases were reported from the United States, Italy, Nigeria, Germany, France, Spain and others (https://www.cdc.gov/vhf/ebola/outbreaks/history/chronology.html).
The life cycle of the Ebola virus gains access to the host via interaction of the virus’ viral glycoprotein with the surface receptors on that of the host cells. The host’s cells then engulf the virus through macropinocytosis pathway. Following endocytosis, the viral membrane then uncoats and fuse with the endosome with the aid of GP1 via the receptor binding domain, while GP2 guides the virus’ fusion (ALL (The lifecycle of the Ebola virus in host cells conclusion) .
Even with this disease high pathogenicity, few clinical trials have succeeded in finding an optimal vaccine and treatment options for humans. Desirable characteristic for an effective drug should have: a high efficacy after a single immunization, long-lasting immunity and rapid-onset of protection (numbered article #4). One of the vaccine that has been tested in non-human primates involves a live, replication-competent recombinant vesicular stomatitis virus (rVSV) where the VSV glycoprotein (GP) gene is replaced by the ZEBOV gp. This vaccine seems promising because it has successfully protected nonhuman primates post-exposure and in clinical trials, a single injection of the vaccine made not only the infected ZEBOV patient immunogenic but also those in close contact with them.
By analyzing human cellular immune responses caused by a single dose of the rVSV-ZEBOV vaccine, Farooq et al. (2016) wanted to show the effect of Tfh antibody titers against the infection. In their study, the authors analyzed the role of Tfh cells as they are important in mediating T-cell dependent humoral immune responses. Few data are available on the relationship between circulating follicular helper T cells (cTfh) and cytokine immune response caused by the rVSV-ZEBOV vaccine. Farooq et al. aimed to give a detailed analysis on the characteristic of the immunological variables sprung forth by this disease.

Methods
Subjects and Samples: The experiment was conducted in conjunction with the Clinical Trials Center at WRAIR. Subjects from WRAIR’s Phase I clinical trials were obtained and peripheral blood mononuclear (PBMC) were collected on the first day and periodically post-vaccination and were then cryopreserved.
Pooling of ZEBOV GP consensus peptides: Lyophilized peptides were purchased from Atlantic Peptides (Lewisburg, PA) and restored with DMSO. Hydrophilic peptides were pooled in equal amounts into 6 aliquots while hydrophobic peptides were excluded.
Human 10-plex Cytokine Pro-Inflammatory Panel: The cryopreserved PBMC of each subject were triggered with the peptide pools for 48 hours on day 0 and day 28 post infection. Meso Scale Discovery’s 10-plex human pro-inflammatory panel kit, a type of assay kit, was then used to measure and analyze the subject’s pro-inflammatory interleukins through the supernatants. Interleukins measured were IL-1ß, IL-8, IL-2, IL-4, IL-6, IL-10, IL-12P70, IL-13, IFN-y, and TNF-a.
Polychromatic flow cytometry staining: This entailed culturing cryopreserved PMBC from days 0, 28 and 56 for 16 hours with a ZEBOV-GP megapool or alone. Anti-human antibodies (CCR6-APC and CD40) were added to the culture at a 1:10 and 1:100 dilution respectively and were later washed and stained with anti-human CD154-biotin, an activation marker and tool for rare cell detection, for 15 minutes. Cells were then incubated with anti-biotin microbeads, washed, incubated with a pre-titrated and optimized antibody cocktail with fluorochrome-conjugated antibodies against CD3-VioBlue, CD4-PerCPVio700, CD185-PEVio770, CD183-VioBrightFITC, CD154-PE and Zombie Aqua Fixable dye for 45 minutes at 4ºC. Cells were enriched and acquired on MACSQuand Analyzer 10.
Gating Strategy: Lymphocytes were gated by scatter and then based on viability and lineage marker CD3 while enriched antigen-specific cells were gated first by the co-expression of CD4 and CD154 followed by the expression of CD4 and CXCR5 cells. Within CD4+CXCR5+ cells, subsets were further identified based on CCR6 and CXCR3 expression as Tfh1, Tfh2, and Tfh17 cells.
Statistical Analysis: Statistical significance between different cohorts and time points were measured using ANOVA while correlation between Tfh frequencies and antibody titers at respective time points were measured by Pearson correlation which was computed and plotted using R software.

Results
Using a Meso Scale Discovery assay, Farooq et al. (2016) was able to identify the cytokine profile as a result of stimulating ZEBOV-GP immune response in PBMC of the subjects. According to Table 2, in response to ZEBVOV-GP, IFN-y was the most secreted cytokine followed by IL-6, IL-8 and IL-12 in all three cohorts. This entails that the cell mediated response is functional because IL-12p70 and IFN- y levels are high, causing low levels of IL-10, an immunosuppressor. T cells need to respond to the infectious agent so had IL-10 levels been high, it would have been an indication of an impaired immune response. The three cohorts were given different intramuscular doses of either 3x〖10〗^6,2x〖10〗^7 and 1x〖10〗^8pfu, respectively.

Using a correlation matrix, the relationship between the cytokines produced in each cohort were measured (FIGURE X). Correlation of a cytokine with itself is 1, thus will both increase simultaneously. In cohort 1, IL-12p70 and IL-13 have a high correlation which is expected. IL-12p70 is a Th1 cytokine which is important in viral infections to induce the cell mediated response while IL-13 is a Th2 cytokine which is like IL-4. It is important for antibody production. Figure X (a) shows a cluster of correlation between IL-10, TNF- a, IL-1b and IL-4 as a result of antigen stimulation. IFN- y does not have any correlation with other cytokines but itself. (B) In cohort two, the strongest positive correlation is seen with TNF- a and IL-4 which is expected since TNF- a is pro-inflammatory and IL-4 is an antibody producing cytokine. Two clusters of cytokines showed correlations: Cluster 1 included IL-2, IL-6, IL-12 and IL-13 while cluster 2 included IL-1B, IL-4, IL-10, and TNF- a. (C) Lastly, cohort 3, IFN- y has only a weak negative correlation with IL-8 and IL-12. This demonstrates that an increase in vaccine dosage change the cytokine signature of the stimulated PBMCs. While the cluster of IL-13, IL-4, IL-10 and TNF- a were present in all three cohorts, cluster of IL-12, IL-2 and IL-13 became present after an increase in dosage as seen in Cohort 2 and Cohort 3. An increase in dose also caused an increase in pro-inflammatory cytokines. Furthermore, a strong negative correlation between the pro-inflammatory cytokine IL-8 and the immunomodulatory IL-6 was detected in all three cohorts. Based on statistical analysis, each cohort differed in the magnitude of IL-2 response (p=.03), IL-6 (p=0.016), TNF- a (p=0.017) and IL-8 (p=0.08).

Farooq et al. (2016) also discovered a significant decrease in the levels of cTFH as a result of immunization with rVSV-ZEBOV-GP. The expression of CD154 on the surfaces of T cells after being exposed to the ZEBOV-GP peptide showed a direct proportional relationship to the dosage. On days 0, 28 and 56, each cohort’s PBMCs were stimulated with overlapping ZEBOV peptide and enhanced based on the expression of the activation marker CD154. This showed an increase in the expression of CD4^+ CXCR5^+ T cells. A significant interaction between time and cohort (p=0.001) indicates that these two variables interact with one another producing an effect.

Figure X. Statistical differences between vaccine cohorts were found on day 28 (p=0.004) and day 56 (p=0.013)

Figure X. Brackets with astericks indicate statistical differences between time points.

Lastly, Farooq et al. (2016) measured the expression the chemokine receptors CXCR3 and CCR6, which are associated with CXCR5 subsets in human peripheral blood, to better understand the impact of the rVSV-ZEBOV vaccine when administered at different doses. This investigation unveiled a heightened presence of cTfh17 cells, followed by cTfh2 cell subset and cTfh1 cell subset, though cTfh17 was the most predominant in cohort 3 when immunized with the rVSV-ZEBOV vaccine. In addition, a significant interaction (p<0.001) for the cTfh17 subpopulation was found upon statistical analysis between time points post-vaccination and cohorts.
Conclusion
All-in-all, as a result of rVSV-ZEBOV vaccination, (1) an increase in the vaccine dose altered the cytokine signature, (2) caused a decrease in cTfh cells in peripheral blood when stimulated with the EBOV peptide and stained for specific CD4+CXCR5+ T and (3), there is a significant increase in cTfh17 subset versus cTfh 2 and cTfh1 cells

Discussion & Conclusion
After vaccination, products of the immunity, such as antigen-specific B cells and Tfh cells remain dormant in the lymph node until a subsequent infection. Follicular T cells are extremely fundamental to the development and differentiation of antigen-specific antibodies. They can be measured by the level of antibodies circulating in the blood post vaccination or infection. Mechanisms that impairs follicular T cells also affect germinal center formation or response which subsequently lead to a decrease of memory B cells and plasma cells that are essential to combat viral infections. Though the germinal center can form in the absence of T cells, the reaction is not long-lasting resulting in diminished plasma cells22.
An effective vaccine is characterized as one that generates high affinity, protective antibodies. The objective of this paper aimed at shedding a light on the effectiveness and the role of Tfh cells in vaccination. Post-vaccination, different cytokines/chemokines are acted upon. In addition, immunization with gp120-encoding DNA provides a clear advantage than immunization gp120 protein alone in providing antibody response. Lastly, the absence of B7-2 directly affects optimal Tfh development and GC B cells.
Antigen specific B cells and Tfh cells work hand-in-hand to promote protective Ab responses. Salek-Ardakani et al. (2012) have evaluated B cells, specifically B7-1 and B7-2, and have demonstrated that B7-2 promote T cells and B cells in Abs responses. They have also shown that B7-2 is required for ideal Bcl6 expression and that B7-2 dependent signal in both B and Tfh cells shape B cell responses. In VACV, in the early response, both B7-1 and B7-2 ligand interaction stimulate signals contributing to favorable CD4 T cells activation and VAVC-specific Th1 differentiation. However, B7-2 interaction with CD28 are crucial for optimal VACV-specific effector CD8 T cell responses. This shows that in the early response in VACV infection, CD4 and CD8 T cells are stimulated by different GC subpopulation for optimal activation. All-in-all, the relative expression of B7-1 and B7-2 on B cells dictate their use. Data demonstrates that B7-2 is continually expressed by B cells and even more so in response to VACV infection. Hence, in the event of a VACV infection, B cells predominantly express B7-2 for interaction with CD28 as they are strongly upregulated.
The role of Tfh cells and GC response have not been clearly defined in prime boost vaccination. Few studies have shown the mechanism in which DNA priming offers a protective advantage. Hollister et al. (2014), have demonstrated, using an HIV gp120 vaccine tactic, the role Tfh cells play in prime boost vaccination. By priming mice with gp120-encoding DNA, a glycoprotein expressed on the surface of HIV cells, and gp120 protein boost vaccination to generate titers with high affinity, they found that priming increased Tfh differentiation and GC B cells and antigen-specific antibody compared with priming with gp120 protein alone. This beneficial increase occurs at earlier time post immunization. Furthermore, several prime-boost HIV-1 vaccines tested in humans with the DNA priming step have shown a significant secretion of Abs versus human trials of HIV-1 without the prime-boost approach. When looking at the number of Th cells which are Tfh cells after DNA priming, there was an increase in the production of CD4 T cells becoming Tfh cells rather than any other subset of Th cells. For B cells, on the other hand, DNA priming enhanced GC memory B cells outcome by prompting the GC to produce more CD73 memory B cells. Lastly, by deleting bcl6 transcription factor, they found that deletion of Bcl6 led to a decrease of Tfh cells differentiation. Together, this data shows effectiveness of DNA priming in vaccination.
Their results indicated a high presence of humoral response with antigen-specific antibody produced by vaccines.
Lastly, Farooq et al. (2016), sought to understand the functionality of CD4+T cells, specifically Tfh cells as they are important for T cell dependent humoral immune response. They first provided a cytokine profile of ZEBOV-glycoprotein-specific immune response in humans. Each cohort were given different dose of the rVSV-ZEBOV vaccine. At a lower dosage, IFN- y was mainly secreted by stimulated cells. As the dosage increased, so did this cytokine and it negatively correlate with IL-8 in cohort 2 and IL-2 in cohort 3. Similar results were obtained by Regules et al. (2015) where antibody titers against ZEBOV glycoprotein were higher in groups receiving higher dosage of the vaccine (20 million PFU and 100 million PFU versus 3 million PFU). Because there were limited amount of patient cells, given the in-exposure of the study subjects to the ZEBOV-GP and the immunization with a single dose of vaccine, Farooq et al. (2016) used a highly sensitive bulk detection method, the Mesoscale platform, to analyze culture supernatants from stimulated PBMCs. They also categorized CD4+T cells based on their expression of markers such as CXCR5, CCR6, and CXCR3. It should be noted that CD4+T cells expressing CCR6 increase in the event of an infection as they are expressed on Th17 cells and play a crucial role in vaccine-induced immune memory. Until recently, Tfh17 cells were associated solely with disease progression but now it’s been known to play a role in protecting against an array of pathogens.
Previous studies have shown that deficiency in CD28 or B7 (B7-1 and B7-2) severely affected Abs response and GC reactions to HSV, vesicular stomatitis, influenza and murine y- herpesvirus. It has also been found that deficiency in B cells after protein immunization, viral infection or parasitic infection in mice causes a defect in Tfh differentiation. This can be corrected by an overexpression of Bcl-6 which aids in lineage differentiation to GC Tfh. Adding the fact that B7-2 interaction with CD28 is essential in maintaining Tfh, suggest that CD28/B7 interaction is important for developing antiviral humoral immune responses. Few studies have yet to show the advantages of DNA priming. Most HIV-1 DNA vaccines have proven futile in eliciting antibody production when the DNA priming step was not added versus incorporating said method.
Though mice are animal models of choice in research, additional insights should be aimed at analyzing Tfh cells and GC B cells in human with prime boost gp120 immunization in HIV-1 vaccine. Moreover, Farooq et al. (2016) experiment was limited to cTfh response. These cells are Tfh-like and were first identified in patients with systemic lupus erythematous and sanroque mice. They express CXCR5, PD-1 and ICOS (Simpson et al., 2010). An increase in cTfh positively correlates with GC and plasma cell response (He et al., 2013 and Bentebibel et al., 2013). Though cTfh cells can be used as a surrogate to GC Tfh cells, Farooq et al. (2016) data in restricted to cTfh cells only. Hence the effectiveness of rVSV-ZEBOV vaccine against the virus was measured in peripheral blood.
Taken together, these studies have shown the importance of follicular T cells in vaccination. Vaccination aids in combatting and reducing infections and disability worldwide. Vaccinations induce antibody responses and are supported by Tfh cells. Absence of Tfh cells or a deficiency of Tfh cells can lead to defective antibody response and disturbance within the germinal center.

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