OBJECTIVE: CpG ODN is a Toll-like receptor 9 agonist with immunotherapeutic potential for many cancer types, including aggressive breast cancers. There is strong interest in utilizing CpG ODN as an adjuvant to improve clinical efficacy of current treatments and immunogenicity of breast cancers not traditionally responsive to active immunotherapy, such as those that are human epidermal growth factor receptor 2 (HER2)-positive. This study aimed to study the efficacy and safety of combination CpG ODN plus anti-HER2 antibody trastuzumab treatment in patients with advanced/metastatic breast cancer.
METHODS: This single-arm, open-label phase II clinical trial treated patients (n = 6) with advanced/metastatic HER2-positive breast cancer with weekly subcutaneous CpG ODN and trastuzumab. Patients may have received any number of prior therapies to be enrolled (most enrolled at median 1 prior line of chemotherapy). Peripheral blood was collected at baseline and weeks 2, 6, 12, and 18 for immune analyses. Six patients were enrolled and 50% achieved stable disease (SD) response.
RESULTS: Median PFS was 8.3 months. Three of the six patients enrolled opted to stop treatment due to tolerability issues. Multiplex assay for cytokine measurements revealed significantly higher VEGF-D levels at week 2 compared to baseline. Peripheral blood mononuclear cells analyzed by flow cytometry showed a significant increase in monocytic MDSC between weeks 6 and 12. Patients with progressive disease tended to have higher levels of week 6 monocytic MDSC and PD-1+ T cells than patients with SD. NK cell populations did not significantly change throughout treatment.
CONCLUSIONS: CpG ODN and trastuzumab treatment of metastatic HER2 + breast cancer was safe but was not tolerable for all patients. This combination did induce potentially predictive immune profile changes in treated patients with metastatic HER2 + breast cancer, the significance of which needs to be further explored.
Why was the study done? Breast cancer that has metastasized (moved outside of the breast and local lymph nodes) is currently considered incurable and can be difficult to treat. Treatments that can stimulate the immune system to recognize cancer cells have been found to be useful for many types of cancers, including some types of breast cancers. This study tested a new immune stimulator (CpG ODN) in combination with a currently on-the-market antibody treatment for breast cancer (trastuzumab). What did the researchers do? The research team enrolled patients who had metastatic breast cancer and treated them all with a combination of trastuzumab and CpG ODN for 12 weeks. These patients were monitored for any side effects/toxicity, monitored for how long their breast cancer responded to this treatment, and monitored for how long they lived after beginning this treatment. Patients also had their blood drawn at different time points to observe how their immune cells and immune proteins (e.g. cytokines) changed on treatment. What did the researchers find? The research team enrolled six patients and found that the treatment was safe and that 50% of the patients treated did not have any breast cancer growth when given CpG ODN plus trastuzumab. Looking at the immune cells in the patient blood samples, some cells that are known to decrease the immune response to cancers (myeloid-derived suppressor cells) did increase towards the end of treatment. What do the findings mean? Overall, CpG ODN and trastuzumab treatment was found to be safe and potentially effective in preventing breast cancer growth.

The CpG oligodeoxynucleotides (CpG ODNs) reportedly possess the capacity to strengthen immunity in mammals. This experiment was conducted to evaluate the impact of dietary supplementation with 17 types of CpG ODNs on intestinal microbiota diversity, antioxidant capacity, and immune-related gene expression profiles of the shrimp Litopenaeus vannamei. Diets including 50 mg kg-1 CpG ODNs wrapped in egg whites were prepared and divided into 17 different groups, with 2 control groups (normal feed and feed with egg whites). These CpG ODNs supplemented diets and the control diets were fed to L. vannamei (5.15 ± 0.54 g) three times daily at 5%-8% shrimp body weight for three weeks. The results of consecutive detection of intestinal microbiota by 16S rDNA sequencing indicated that 11 of the 17 types of CpG ODNs significantly enhanced intestinal microbiota diversity, increased the populations of several probiotic bacteria, and activated possible mechanisms relevant to diseases. The immune-related genes expression and antioxidant capacity in hepatopancreas further demonstrated that the 11 types of CpG ODNs effectively improved the innate immunity of shrimp. Additionally, histology results showed that the CpG ODNs in the experiment did not damage the tissue structure of hepatopancreas. The results suggest that CpG ODNs could be used as a trace supplement to improve the intestinal health and immunity of shrimp.

The neonatal immune system is distinct from the immune system of older individuals rendering neonates vulnerable to infections and poor responders to vaccination. Adjuvants can be used as tools to enhance immune responses to co-administered antigens. Antibody (Ab) persistence is mediated by long-lived plasma cells that reside in specialized survival niches in the bone marrow, and transient Ab responses in early life have been associated with decreased survival of plasma cells, possibly due to lack of survival factors. Various cells can secrete these factors and which cells are the main producers is still up for debate, especially in early life where this has not been fully addressed. The receptor BCMA and its ligand APRIL have been shown to be important in the maintenance of plasma cells and Abs. Herein, we assessed age-dependent maturation of a broad range of bone marrow accessory cells and their expression of the survival factors APRIL and IL-6. Furthermore, we performed a comparative analysis of the potential of 5 different adjuvants; LT-K63, mmCT, MF59, IC31 and alum, to enhance expression of survival factors and BCMA following immunization of neonatal mice with tetanus toxoid (TT) vaccine. We found that APRIL expression was reduced in the bone marrow of young mice whereas IL-6 expression was higher. Eosinophils, macrophages, megakaryocytes, monocytes and lymphocytes were important secretors of survival factors in early life but undefined cells also constituted a large fraction of secretors. Immunization and adjuvants enhanced APRIL expression but decreased IL-6 expression in bone marrow cells early after immunization. Furthermore, neonatal immunization with adjuvants enhanced the proportion of plasmablasts and plasma cells that expressed BCMA both in spleen and bone marrow. Enhanced BCMA expression correlated with enhanced vaccine-specific humoral responses, even though the effect of alum on BCMA was less pronounced than those of the other adjuvants at later time points. We propose that low APRIL expression in bone marrow as well as low BCMA expression of plasmablasts/plasma cells in early life together cause transient Ab responses and could represent targets to be triggered by vaccine adjuvants to induce persistent humoral immune responses in this age group.

BACKGROUND: Cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) (K3)-a novel synthetic single-stranded DNA immune adjuvant for cancer immunotherapy-induces a potential Th1-type immune response against cancer cells. We conducted a phase I study of CpG ODN (K3) in patients with lung cancer to assess its safety and patients\' immune responses.
METHODS: The primary endpoint was the proportion of dose-limiting toxicities (DLTs) at each dose level. Secondary endpoints included safety profile, an immune response, including dynamic changes in immune cell and cytokine production, and progression-free survival (PFS). In a 3 + 3 dose-escalation design, the dosage levels for CpG ODN (K3) were 5 or 10 mg/body via subcutaneous injection and 0.2 mg/kg via intravenous administration on days 1, 8, 15, and 29.
RESULTS: Nine patients (eight non-small-cell lung cancer; one small-cell lung cancer) were enrolled. We found no DLTs at any dose level and observed no serious treatment-related adverse events. The median observation period after registration was 55 days (range: 46-181 days). Serum IFN-α2 levels, but not inflammatory cytokines, increased in six patients after the third administration of CpG ODN (K3) (mean value: from 2.67 pg/mL to 3.61 pg/mL after 24 hours). Serum IFN-γ (mean value, from 9.07 pg/mL to 12.7 pg/m after 24 hours) and CXCL10 levels (mean value, from 351 pg/mL to 676 pg/mL after 24 hours) also increased in eight patients after the third administration. During the treatment course, the percentage of T-bet-expressing CD8+ T cells gradually increased (mean, 49.8% at baseline and 59.1% at day 29, p = 0.0273). Interestingly, both T-bet-expressing effector memory (mean, 52.7% at baseline and 63.7% at day 29, p = 0.0195) and terminally differentiated effector memory (mean, 82.3% at baseline and 90.0% at day 29, p = 0.0039) CD8+ T cells significantly increased. The median PFS was 398 days.
CONCLUSIONS: This is the first clinical study showing that CpG ODN (K3) activated innate immunity and elicited Th1-type adaptive immune response and cytotoxic activity in cancer patients. CpG ODN (K3) was well tolerated at the dose settings tested, although the maximum tolerated dose was not determined.
BACKGROUND: UMIN-CTR number 000023276. Registered 1 September 2016, https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000026649.

A range of safe and effective vaccines against SARS CoV 2 are needed to address the COVID 19 pandemic. We aimed to assess the safety and efficacy of the COVID-19 vaccine SCB-2019.
This ongoing phase 2 and 3 double-blind, placebo-controlled trial was done in adults aged 18 years and older who were in good health or with a stable chronic health condition, at 31 sites in five countries (Belgium, Brazil, Colombia, Philippines, and South Africa). The participants were randomly assigned 1:1 using a centralised internet randomisation system to receive two 0·5 mL intramuscular doses of SCB-2019 (30 μg, adjuvanted with 1·50 mg CpG-1018 and 0·75 mg alum) or placebo (0·9% sodium chloride for injection supplied in 10 mL ampoules) 21 days apart. All study staff and participants were masked, but vaccine administrators were not. Primary endpoints were vaccine efficacy, measured by RT-PCR-confirmed COVID-19 of any severity with onset from 14 days after the second dose in baseline SARS-CoV-2 seronegative participants (the per-protocol population), and the safety and solicited local and systemic adverse events in the phase 2 subset. This study is registered on EudraCT (2020-004272-17) and ClinicalTrials.gov (NCT04672395).
30 174 participants were enrolled from March 24, 2021, until the cutoff date of Aug 10, 2021, of whom 30 128 received their first assigned vaccine (n=15 064) or a placebo injection (n=15 064). The per-protocol population consisted of 12 355 baseline SARS-CoV-2-naive participants (6251 vaccinees and 6104 placebo recipients). Most exclusions (13 389 [44·4%]) were because of seropositivity at baseline. There were 207 confirmed per-protocol cases of COVID-19 at 14 days after the second dose, 52 vaccinees versus 155 placebo recipients, and an overall vaccine efficacy against any severity COVID-19 of 67·2% (95·72% CI 54·3-76·8), 83·7% (97·86% CI 55·9-95·4) against moderate-to-severe COVID-19, and 100% (97·86% CI 25·3-100·0) against severe COVID-19. All COVID-19 cases were due to virus variants; vaccine efficacy against any severity COVID-19 due to the three predominant variants was 78·7% (95% CI 57·3-90·4) for delta, 91·8% (44·9-99·8) for gamma, and 58·6% (13·3-81·5) for mu. No safety issues emerged in the follow-up period for the efficacy analysis (median of 82 days [IQR 63-103]). The vaccine elicited higher rates of mainly mild-to-moderate injection site pain than the placebo after the first (35·7% [287 of 803] vs 10·3% [81 of 786]) and second (26·9% [189 of 702] vs 7·4% [52 of 699]) doses, but the rates of other solicited local and systemic adverse events were similar between the groups.
Two doses of SCB-2019 vaccine plus CpG and alum provides notable protection against the entire severity spectrum of COVID-19 caused by circulating SAR-CoV-2 viruses, including the predominating delta variant.
Clover Biopharmaceuticals and the Coalition for Epidemic Preparedness Innovations.

The mechanisms explaining excess morbidity and mortality in respiratory infections among males are poorly understood. Innate immune responses are critical in protection against respiratory virus infections. We hypothesised that innate immune responses to respiratory viruses may be deficient in males. We stimulated peripheral blood mononuclear cells from 345 participants at age 16 years in a population-based birth cohort with three live respiratory viruses (rhinoviruses A16 and A1, and respiratory syncytial virus) and two viral mimics (R848 and CpG-A, to mimic responses to SARS-CoV-2) and investigated sex differences in interferon (IFN) responses. IFN-α responses to all viruses and stimuli were 1.34-2.06-fold lower in males than females (P = 0.018 -  < 0.001). IFN-β, IFN-γ and IFN-induced chemokines were also deficient in males across all stimuli/viruses. Healthcare records revealed 12.1% of males and 6.6% of females were hospitalized with respiratory infections in infancy (P = 0.017). In conclusion, impaired innate anti-viral immunity in males likely results in high male morbidity and mortality from respiratory virus infections.

This article provides a detailed procedure for the chemical synthesis and characterization of photoswitchable hydrazone phosphoramidite and its incorporation into oligodeoxynucleotides. The synthesis starts with commercially available deoxyuridine, followed by conversion of the 4-oxo into a 4-chloro moiety via Appel reaction to install the key hydrazone group in the absence of base. The hydrazone phosphoramidite building block is compatible with the conventional amidite chemistry protocols for solid-phase synthesis of oligodeoxynucleotides. Our method expands the current nucleotide pool by adding a novel, functional DNA building block that is suitable for a broad spectrum of applications, including the regulation of DNA-enzyme interactions and DNA synthesis by irradiation with cell-friendly blue light. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of photoswitchable hydrazone phosphoramidite Basic Protocol 2: Synthesis and purification of oligodeoxynucleotides containing the hydrazone photoswitch Basic Protocol 3: Primer extension assay for functionality studies of hydrazone cytidine.

MVC-COV1901, a recombinant protein vaccine containing pre-fusion-stabilised spike protein S-2P adjuvanted with CpG 1018 and aluminium hydroxide, has been shown to be well tolerated with a good safety profile in healthy adults aged 20-49 years in a phase 1 trial, and provided a good cellular and humoral immune responses. We present the interim safety, tolerability, and immunogenicity results of a phase 2 clinical trial of the MVC-COV1901 vaccine in Taiwan.
This is a large-scale, double-blind, randomised, placebo-controlled phase 2 trial done at ten medical centres and one regional hospital in Taiwan. Individuals aged 20 years or older who were generally healthy or had stable pre-existing medical conditions were eligible for enrolment. Exclusion criteria included (but were not limited to) travel overseas within 14 days of screening, intention to travel overseas within 6 months of the screening visit, and the absence of prespecified medical conditions, including immunosuppressive illness, a history of autoimmune disease, malignancy with risk to recur, a bleeding disorder, uncontrolled HIV infection, uncontrolled hepatitis B and C virus infections, SARS-CoV-1 or SARS-CoV-2 infections, an allergy to any vaccine, or a serious medical condition that could interfere with the study. Study participants were randomly assigned (6:1) to receive two doses of either MVC-COV1901 or placebo, administered via intramuscular injection on day 1 and day 29. MVC-COV1901 contained 15 μg of S-2P protein adjuvanted with 750 μg CpG 1018 and 375 μg aluminium hydroxide in a 0·5 mL aqueous solution, and the placebo contained the same volume of saline. Randomisation was done centrally by use of an interactive web response system, stratified by age (≥20 to <65 years and ≥65 years). Participants and investigators were masked to group assignment. The primary outcomes were to evaluate the safety, tolerability, and immunogenicity of MVC-COV1901 from day 1 (the day of the first dose) to day 57 (28 days after the second dose). Safety was assessed in all participants who received at least one dose. Immunogenicity was assessed by measuring geometric mean titres (GMTs) and seroconversion rates of neutralising antibody and antigen-specific IgG in the per-protocol population. This study is registered with ClinicalTrials.gov, NCT04695652.
Of 4173 individuals screened between Dec 30, 2020, and April 2, 2021, 3854 were enrolled and randomly assigned: 3304 to the MVC-COV1901 group and 550 to the placebo group. A total of 3844 participants (3295 in the MVC-COV1901 group and 549 in the placebo group) were included in the safety analysis set, and 1053 participants (903 and 150) had received both doses and were included in the per-protocol immunogenicity analysis set. From the start of this phase 2 trial to the time of interim analysis, no vaccine-related serious adverse events were recorded. The most common solicited adverse events in all study participants were pain at the injection site (2346 [71·2%] of 3295 in the MVC-COV1901 group and 128 [23·3%] of 549 in the placebo group), and malaise or fatigue (1186 [36·0%] and 163 [29·7%]). Fever was rarely reported (23 [0·7%] and two [0·4%]). At 28 days after the second dose of MVC-COV1901, the wild-type SARS-CoV-2 neutralising antibody GMT was 662·3 (95% CI 628·7-697·8; 408·5 IU/mL), the GMT ratio (geometric mean fold increase in titres at day 57 vs baseline) was 163·2 (155·0-171·9), and the seroconversion rate was 99·8% (95% CI 99·2-100·0).
MVC-COV1901 has a good safety profile and elicits promising immunogenicity responses. These data support MVC-COV1901 to enter phase 3 efficacy trials.
Medigen Vaccine Biologics and Taiwan Centres for Disease Control, Ministry of Health and Welfare.

Natural products have failed to meet the urgent need for drug discovery in recent decades due to limited resources, necessitating new strategies for re-establishing the key role of natural products in hit screening. This work introduced DNA-encoding techniques into the synthesis of phenolic acid-focused libraries containing 32 000 diverse compounds. Online selection of the library using immobilized angiotensin II type I receptor (AT1R) resulted in seven phenolic acid derivatives. The half-maximal concentration (IC50) of hit 1 for the right shift of the [125I]-Sar1-AngII competition curve was 19.6 nM. Pharmacological examination of renovascular hypertensive rats demonstrated that hit 1 significantly lowered the blood pressure of the animals without changing their heart rates. These results were used to create a general strategy for rapid and unbiased discovery of hits derived from natural products with high throughput and efficiency.

Cobitolimod is a topically administered, DNA-based oligonucleotide that activates Toll-like receptor 9 (TLR9), and previous research has shown clinical efficacy in patients with moderate-to-severe ulcerative colitis. Here we assessed the efficacy and safety of different dose regimens of cobitolimod for induction therapy in patients with moderate-to-severe, left-sided ulcerative colitis.
CONDUCT was a randomised, double-blind, five-arm, placebo-controlled, dose-ranging phase 2b study that recruited patients with moderate-to-severe, left-sided ulcerative colitis, with inadequate response to conventional or biological therapies, from 91 hospitals or outpatient clinics in 12 European countries. Eligible patients had a Mayo score of 6-12 with a centrally read endoscopic subscore (modified to exclude friability from grade 1) of 2 or higher and no individual subscore of less than 1, and confirmation of left-sided disease. Patients were randomised (1:1:1:1:1; block size of ten) via a computer-generated schedule and centralised interactive voice and web response system to receive rectal enemas of cobitolimod at 31 mg, 125 mg, or 250 mg at weeks 0 and 3 (2 × 31 mg, 2 × 125 mg, and 2 × 250 mg groups), cobitolimod at 125 mg at weeks 0, 1, 2, and 3 (4 × 125 mg group), or placebo. Randomisation was stratified by current glucocorticosteroid and previous tumour necrosis factor inhibitor treatment. Patients and all study personnel were masked to treatment allocation. The primary endpoint was the proportion of patients achieving clinical remission (Mayo subscores for rectal bleeding of 0, for stool frequency of 0 or 1 [with ≥1-point decrease from baseline], and for endoscopy of 0 or 1 [excluding friability]) at week 6. The primary analysis (based on intention to treat) and safety analysis were done in all randomly assigned patients who received at least one dose of active study drug or placebo. In this exploratory study, statistical tests were one-sided; p values of less than 0·10 were regarded as statistically significant, with no adjustment for multiplicity. This study is registered with ClinicalTrials.gov, NCT03178669, and is completed; the results here represent the final analysis.
213 patients were randomly assigned between June 30, 2017, and June 26, 2019. Of these, 211 patients received study treatment: 40 in the cobitolimod 2 × 31 mg group, 43 in the 2 × 125 mg group, 42 in the 4 × 125 mg group, 42 in the 2 × 250 mg group, and 44 in the placebo group. A greater proportion of patients were in clinical remission at week 6 in the cobitolimod 2 × 250 mg group than in the placebo group (nine [21%] of 42 patients vs three [7%] of 44; odds ratio [OR] 3·8 [80% CI 1·5-9·5]; one-sided p=0·025). We identified no significant difference in the proportion of patients with clinical remission in the cobitolimod 2 × 31 mg group (five [13%] of 40 patients; OR 2·0 [80% CI 0·7-5·5], p=0·18), 2 × 125 mg group (two [5%] of 43; 0·7 [0·2-2·2], p=0·66), or 4 × 125 mg (four [10%] of 42; 1·4 [0·5-3·9], p=0·33) compared with the placebo group. Treatment-emergent adverse events occurred in 21 (48%) patients in the placebo group, ten (25%) patients in the cobitolimod 2 × 31 mg group, 17 (40%) patients in the 2 × 125 mg group, 15 (36%) patients in the 4 × 125 mg group, and 18 (43%) patients in the 2 × 250 mg group. Severe adverse events occurred in eight (4%) of 211 patients (worsening of ulcerative colitis [seven patients] and abdominal hernia and wound dehiscence [one patient]). Ten patients (two [5%] in the placebo group, two [5%] in the cobitolimod 2 × 31 mg group, two [5%] in the 4 × 125 mg, and four [10%] in the 2 × 250 mg group) had a total of 13 serious adverse events; these were worsening of ulcerative colitis (eight events) and pruritus, rash, abdominal hernia, fascia dehiscence, and deep vein thrombosis (one event each). One patient in the placebo group died from total organ failure after receiving a colectomy for a serious adverse event of disease worsening.
Two topical administrations of cobitolimod 250 mg were well tolerated and more effective than placebo in inducing clinical remission 6 weeks after the start of treatment. TLR9 activation is a promising novel therapeutic target in ulcerative colitis and warrants further testing, with phase 3 trials of cobitolimod planned.
InDex Pharmaceuticals.