2.2. Immunization Protocol and Sample Collection

After agreeing to participate based on written informed consent, a blood sample was taken for biochemical and hematological measurements. Clinical assessment, including anthropometric measurements and physical examination, was performed during the baseline visit. The volunteers were informed that the study would be investigating the effects on immune responses to PCV13 vaccine of a probiotic mix containing Lactobacillus plantarum DSM24730(r), Streptococcus thermophilus DSM24731(r), Bifidobacterium breve DSM24732(r), L. paracasei DSM24733(r), L. delbrueckii subsp. bulgaricus DSM24734(r), L. acidophilus DSM24735(r), B. longum DSM24736(r), and B. infantis DSM24737(r) available as Vivomixx^® (Mendes S.A., Switzerland) in Europe and Visbiome^® in USA. Enrolled HIV+ subjects were randomized and divided into two groups: 40% of volunteers received a placebo dose (Group 1), while 60% received two + two sachets a day (each sachet contains 450 billion CFU, total dose per day 1800 billion CFU) of Vivomixx^® (Group 2). The product supplementation took place four weeks before the control visit. At the control visit, a single dose of PCV13 vaccine was administered intramuscularly in the deltoid region. There were no cases of pneumococcal disease within one month post-immunization. Both groups were subjected to blood sampling, which involved 10 mL being collected using BD Vacutainer^TM tubes with sodium heparin (BD Biosciences, Baltimore, MD, USA) at the basal visit (T0), after four weeks of supplementation and immediately before immunization (T1), and after eight weeks of supplementation and four weeks post-immunization (T2). Serum obtained after centrifugation of peripheral blood was stored at −80 °C until use. Whole unstimulated saliva samples were also collected at each visit. About five mL of unstimulated whole saliva was collected from each subject in sterile polypropylene tubes. The samples were centrifuged at 10,000× g for 15 min at 4 °C and the supernatant was immediately aliquoted and frozen (−80 °C), until being analyzed (Figure 1). The study protocol was approved by the Ethics Committee at the University “G. d’Annunzio” Chieti-Pescara (Ethics Committee Project No. 20, 17 October 2019) and was performed under the ethical standards laid down in the 1964 Declaration of Helsinki.

Study design flow chart. Graphical description of randomized sampling.