a micro-sterile inflammation array as an adjuvant for influenza vaccines - skin resurfacing at home

There is an urgent need for skin vaccination.
Here we report Micro
Sterile inflammation induced by vaccination sites can enhance the immune response of animal models to influenza vaccines.
Hand-held non-vaccination sites
The ablation fraction laser before the administration of the vaccine skin produced a series of self-contained
Healing area (MTZs)in the skin.
Dead cells in MTZs send "dangerous" signals that attract a large number of antigens
Presenting cells, especially plasma-like dc cells (pDCs)
Form a micro around each MTZ
Sterile inflammatory array.
Key role of PDCs in adaptability significant weakening of immunity after systemic exhaustion of pDCs, local application of TNF-
Alpha Inhibitor or zero mutation of interferon regulatory factor 7 (IRF7).
Compared to traditional supplements that cause persistent inflammation and skin damage, micro-
Aseptic inflammation enhances the efficacy of the influenza vaccine, but the adverse reactions are reduced.
A 6-8-week-old, close-to-B/c mouse and a forward-to-Swiss Webster mouse were purchased from the Charles River Laboratory.
The mice of the two sex types were randomly used with no significant difference. Eighteen-month-
Old mice (old mice)
Purchased from the National Institute of Aging (NIA).
Irf-/-mice with a background of 57-/-mice are a gift from Dr. T.
A control of Taniguchi and mice from the University of Tokyo was obtained from the Jackson Lab. MHC II-
Green fluorescent protein mice expressing class II molecules are injected with enhanced green fluorescent protein, a gift for dr Boes and Ploegh at Harvard Medical School.
The four-month-old male Yorkshire pig was obtained from the teaching and research resources of the University of taffz.
These animals live in specific pathogens.
Free animal facilities at Massachusetts General Hospital (MGH)
Meet the guidelines for institutions, hospitals and NIH.
All studies have been reviewed and approved by the MGH Institutional Animal Care and Use Committee. A FDA-approved, home-
Using a NAFL laser in mice (
PaloVia Skin update laser, Palomar medical technology).
Handheld devices issued 1,410-
Two scans using nm laser and medium power produce overlapping MTZs at the inoculation point.
Fraxel SR-1 treatment for pigs1500 laser (Solta Medical).
This clinical device emits a laser array with a coverage of 17%, each passing 93 mtzscm cm and 35 mj per Microbeam.
Get pandemic A/California/7/2009 H1N1 influenza virus from US culture collection (ATCC, FR-201).
The virus is 10-day-
Old embryonic eggs at 35 °c for 3 days, collected by speeding centrifuge, frozen at 80 °c until used.
The number of tissue culture infection doses of 50% were measured (TCID)
In dog kidney cells (ATCC, CCL-34).
To challenge mice, the virus adapted to three cycles of nasal drip-
The preparation of lung homogenates and the resulting virus infection test tested the death rate of 50% (LD)
In adult male/c mice following the standard protocol.
California/7/2009 influenza A (H1N1) vaccine (
Sanofi Buster)
Obtained from MGH pharmacy and BEI Resources, at 0.
Unless otherwise indicated, 06 μg HA per mouse or 3 μg HA per pig.
The mice to be immune were removed from the hair on the lower back skin, vaccinated with the flu vaccine ID the next day, or laser ironed with the ID before vaccination.
After ID immunization, the vaccination site is either placed separately or applied locally with IMIQ (
Aldara, 3 CUCM pharmaceutical company).
The inoculation site is then covered with 3 m Tegaderm film to protect it.
Get me the flu vaccine, squalene-AddaVax. based nano-
Emulsion accessories (Invivogen)
Mix with the flu vaccine in a 1:1 ratio and inject IM with a total volume of 20 μ l.
Temperature control devices monitor the body temperature of some mice every hour (FHC).
Determination of blood cytokines by enzyme
Immune adsorption test (ELISA)kit (eBiosciences).
Dripping into the nose of 10 × LD mice into the immune mice and control mice-
Adapt to the 2009 H1N1 virus.
Unless otherwise specified, monitor the weight and survive for 14 days per day.
In some infection experiments, lungs were collected 4 days after the challenge, and lung virus titer was determined by TCID.
Animals were injected with telazol to immunize pigs (2. 2u2009mgu2009kg)/xylazine (2. 2u2009mgu2009kg)/atropine (0. 04u2009mgu2009kg)
And maintained at the end of the day (2–3%)
Inhalation During hair removal and immunization.
The immune procedure is similar to that of mice vaccinated with the 100 influenza μ l (3 μg HA content)
Vaccinate into the outer hind leg skin alone or in the presence of NAFL, IMIQ or NAFL/imiq aids.
In order to quantify the local skin reaction, shoot the inoculation site and circle and analyze the erythema area of each inoculation site through Image Pro Premier software (
Media Control, company)
Calculate the average erythema area of each inoculation site three times.
Detect HAI drops according to the published scheme (PMID: 19274084).
Serum samples incubated with receptors
Enzyme destruction (Denka Seiken)
Overnight at 37 °c and then heat-inactivated for 30 min at 56 °c.
The obtained serum samples were incubated with four HA unit influenza viruses at 37 °c for 1 hour after continuous dilution and then 0.
5% chicken red blood cells (
Charles River Laboratory
30 minutes at room temperature.
HAI titer is defined as the reciprocal of the highest dilution that inhibits HA. Vaccine-
Specific IgG, IgG1, IgG2a and IgA antibodies were determined by ELISA.
In short, in NaHCO buffer, 1 μg ml of recombinant HA was applied to the ELISA plate, ph9. 6.
After incubation with continuously diluted serum samples
Sheep resistancemouse IgG (
NA931V, dilution 1:6, 000, GE Healthcare), IgG1 (A90-
105 P, Bethel, diluted 1:10), IgG2a (61-
0220, Life Technology, dilution 1:2, 000)or IgA (A90-
Bethyl, 103 P, 1: 10)
Antibodies were added to measure specific subtypes.
For the mice,mouse IgG2c (1079-
05, South biotechnology, 1:5, 000)
Replace anti-with antibodies-IgG2a antibody.
One week after immunization, blood samples from immune mice and control mice were collected into heparin tubes through tail vein bleeding.
After the red blood cells were lysed, the peripheral blood single nuclear cells were isolated.
Single nuclear cells in peripheral blood (10 cells per ml)
Co-incubation with influenza vaccine (
1 μg HA ml HA content)and anti-CD28 (clone 37.
51, BD Pharmingen, 4 u2009 μg ml)
Antibody overnightGolgi-Plug (BD Pharmingen)
Added at the last 5 Thanh of incubation to prevent the secretion of cytokines.
Stimulated cells are stained with fluorescence
Binding antibody against CD4 (clone RM4-
5, Biolegend, diluted 1:100), CD8 (clone 53-6.
7. Biolegend, dilution 1:200)and IFNγ (clone XMG1.
Month, Biolegend, 1:100)
, And then flow cell analysis as described earlier.
In the presence of the NAFL/IMIQ vaccine, mice were vaccinated with a flu vaccine or with a vaccine mixed with AddaVax.
The tissue of the inoculation site was anatomical at the specified time, fixed and stained with the standard sumosperm-ihong procedure.
After 3 days of immunization, a similar histological examination was performed in the pig.
Scanning and analysis of slides by Nano zoomer (Hamamatsu).
Ears of the mhii-
The treatment of the new Armed Forces of Liberia or the new Armed Forces of Liberia/IMIQ was carried out by the genetically modified mouse.
Green fluorescent protein cells in the skin and genuine leather through in vivo imaging
Photon focal microscopy (Olympus FV-1000). Three-
Through Image J software, D reconstruction is used to visualize the accumulation of green fluorescent cells around a single MTZs.
Ears of the mhii-
In the presence of the specified adjuvant, green fluorescent protein transgenic mice were vaccinated with influenza vaccine.
The outer ear flap was prepared at the specified time after immunization, fixed with 4% formaldehyde for the night at 4 °c, and closed with 2% fetal bovine serum/PBS at room temperature for 2 u2009 h.
Fixed samples against rat-LYVE-1 (
Clone 223322, R & D system, 8 μ g ml)
Identification of antibodies to lymphatic vessels and rabbit antibodiesCollagen IV (
Ab6586, Abcam, 10 μg ml)
Antibodies to lymph and blood vessels were marked overnight at 4 °c and then stained with Cy3-Combinedrat (
Life Technology, diluted 1:100)and NL637-Combinedrabbit (
NL005, R & D system, dilution 1:100)
Antibody for overnight at 4 °c.
The dyed samples were installed and imaged by two people
Photon focal microscopy (Olympus FV-1000).
The lower back skin of the mouse was exposed to the NAFL/IMIQ supplement. Twenty-
After 4 hours, remove the full thick skin of the laser irradiation site, prepare the frozen tissue slices, and use the-Siglec H mouse (
EBioscience clone RECC, 10 μg ml)
Or same type of control antibody stained with DL594-
Sheep resistancerat IgG (SA5-
10081, hot puncture, dilution 1:100)antibody.
Slides are loaded with a loading medium containing 4', 6-nuclear stained fluorescent dyediamidino-2-
Imaging by a co-Focusing microscope.
Cut, chop, and digest the skin of the inoculation site with Dispase II (
Life Technology)
Plate bacteria D (Roche)
For 2 h at 37 °c
Digested skin tissue through 100
μ m unit filter for preparation of single battery filter
Cell suspension
Results anti-cell-
CD16/CD32 antibody (
Clone 93, dilute 1:50)
20 minutes, then stained with the following fluorescence-
Binding antibody for 30 minutes on ice: APC-anti–PDCA-1(
Clone 927, dilute 1:300), PE-anti-CD11c (
Cloning N418, ebioscience, dilution 1:100), BV421-anti-CD11b (
Clone M1/70, dilute 1:100), FITC-anti-MHCII (clone M5/114. 15.
2. dilution 1:200), FITC-anti-Ly6C (clone HK1.
4. dilution 1:200), and APC/Cy7-anti-B220 (clone RA3-
6B2, diluted 1:100).
All antibodies are purchased from Biolegend unless otherwise stated.
The stained cells were obtained on FACSAria (BD)
Analysis using FlowJo software (Tree Star).
Anti-with two doses-mPDCA-1 antibody (clone JF05-1C2. 4.
1. mi Teng Yi biotechnology)
Before immunization, 400 μg per dose was used at 24 and 0 µh respectively.
Flow microscopy of pDCs in blood samples confirmed exhaustion efficiency.
After one week, the effect of dc deficiency on cellular immune response was evaluated and the body fluid immune response was analyzed two weeks after immunization.
Collect and order dLNs
Preparation, counting and staining of cell suspension-anti-CD11c (
Cloning N418, ebiocience, dilution 1:100), APC-anti-CD40 (
Clone 3/23, Biolegend, diluted 1:100)and PerCp-Cy5. 5-anti-CD86 (clone GL-
1, Biolegend, diluted 1:100)
Antibody, then flow cell.
Based on the total number of cells in the lymph nodes and the percentage of each subset of cells, the total number of CD11c cells, cd4011c cells or CD11c cells in each lymph node is calculated.
To analyze the expression of cytokines and inflammatory cytokines at the inoculation site, the full thickness of the skin area of about 7 × 10mm was removed, total RNA was extracted, reverse
Use the SYBR Green PCR kit (Roche).
GAPDH is used as internal control.
All genes and their primers are listed in. Two-tailed -test (-test)
Used to analyze the differences between the two groups, one group
ANOVA was used across multiple groups.
Bonferroni correction after ANOVA (
Nova/Bonferroni)
Used to compare selected pairs, dun is followed by a test by Dunnett (ANOVA/Dunnett's)
All groups were compared with the control group. -
Calculate the value with PRISM Software (GraphPad)
And one is considered a major difference-
The value is less than 0. 05.
Sample size was designed based on preliminary experiments to give statistical capability.
No animals were excluded from the analysis.
The researchers did not turn a blind eye to these experiments, which were carried out under highly standardized and predefined conditions, except for the photo analysis performed in the researchers and the SU Mujing-Yi red slideblind fashion.