Vunipola picked up a knee injury in club side Saracens' 41-13 victory over Sale Sharks at Allianz Park on Saturday afternoon.
The Red Rose squad are due to meet in Oxford today for the start of a three-day training camp ahead of Autumn Internationals against Argentina, Australia and Samoa.
Harlequins ace Clifford will now be among the 33-man group, with Vunipola expected to spend an extended time on the sidelines.
Updated England training squad:
Jack Clifford (Harlequins), Dan Cole (Leicester Tigers), Tom Curry (Sale Sharks), Tom Dunn (Bath Rugby), Charlie Ewels (Bath Rugby), Ellis Genge (Leicester Tigers), Jamie George (Saracens), Dylan Hartley (Northampton Saints), Nathan Hughes (Wasps), Nick Isiekwe (Saracens), Maro Itoje (Saracens), Joe Launchbury (Wasps), Courtney Lawes (Northampton Saints), Joe Marler (Harlequins), Chris Robshaw (Harlequins), Sam Underhill (Bath Rugby), Harry Williams (Exeter Chiefs), Mako Vunipola (Saracens)
Mike Brown (Harlequins), Danny Care (Harlequins), Elliot Daly (Wasps), Owen Farrell (Saracens), George Ford (Leicester Tigers), Piers Francis (Northampton Saints), Alex Lozowski (Saracens), Jonny May (Leicester Tigers), Jack Nowell (Exeter Chiefs), Henry Slade (Exeter Chiefs), Marcus Smith (Harlequins), Ben Te'o (Worcester Warriors), Anthony Watson (Bath Rugby), Marland Yarde (Harlequins), Ben Youngs (Leicester Tigers)